Antibiotics and methods for manufacturing the same

ABSTRACT

The present invention generally relates to a novel highly discriminating antibiotic, plantazolicin, a plantazolicin-like compound and to pharmaceutical compositions comprising the same. Also provided are methods for producing and using plantazolicin. Due to its bactericidal activity against  Bacillus anthracis , plantazolicin and plantazolicin-like compounds can be used in methods for treating and/or preventing anthrax infections.

FIELD OF THE INVENTION

The present invention generally relates to a novel highly discriminatingantibiotic, plantazolicin (PZN), which was isolated from Bacillusamyloliquefaciens FZB42 or Bacillus pumilus, and to pharmaceuticalcompositions comprising plantazolicin or a salt or an ester thereof.Also provided are methods for producing and using such plantazolicincompounds.

BACKGROUND OF THE INVENTION

With facile access to low-cost next-generation DNA sequencingtechnology, there has been a recent surge in genome sequencing. Theavailability of nearly 2,000 microbial genomes has rekindled interest inthe biosynthetic capabilities of bacteria (Challis, G. L. (2008) J MedChem 51, 2618-2628; Gross, H. (2009) Curr Opin Drug Discov Devel 12,207-219; Melby et al., (2011) Curr Opin Chem. Biol., 15(3):369-78).Given the status of natural products and their derivatives as thelargest source of all medicines, exploring uncharted biosyntheticterritory holds vast potential (Newman and Cragg, (2007) J Nat Prod 70,461-477). One such region of natural product space includes thethiazole/oxazole-modified microcin (TOMM) family (Haft et al., (2010)BMC Biol 8, 70; Lee et al., (2008) Proc Natl Acad Sci USA 105,5879-5884; Scholz et al. (2011) J Bacteriol 193, 215-224).

Microcins are antibacterial peptides that differ from popularbroad-range antibiotics in a variety of ways. One important differenceis that microcins target a narrow spectrum of bacteria. As a result,natural human microbial flora will go undisturbed aiding in decreasedside effects. A second important difference is that microcins are lesslikely to be horizontally transferred due to their narrow targetspectrum and complex machinery required for synthesis and export, whichis often encoded on multiple genes.

Unlike the well-known non-ribosomal peptides and polyketides, TOMMs arederived from inactive, ribosomally synthesized precursor peptides. EachTOMM precursor peptide harbors an N-terminal leader region that servesas the binding site for enzymes that posttranslationally modify aC-terminal core region (Madison et al., (1997) Mol Microbiol 23,161-168; Mitchell et al., (2009) J Biol Chem 284, 13004-13012). Thedistinguishing chemical features of a TOMM are heterocycles that derivefrom cysteine, serine, and threonine residues, which are abundant in thecore region of the precursor peptide. During processing by a geneticallyconserved cyclodehydratase, select cysteines and serine/threonine aminoacids undergo peptide backbone cyclization to become thiazoline and(methyl)oxazoline heterocycles. A subset of these are further subjectedto a flavin mononucleotide (FMN)-dependent dehydrogenation, which yieldsthe aromatic thiazole and (methyl)oxazole heterocycles. The formation ofheterocycles on TOMM precursor peptides is dependent on the presence ofa third component, termed the docking protein, whose exact functionremains enigmatic (McIntosh, J. A. and Schmidt, E. W., (2010)Chembiochem 11, 1413-1421; Milne et al., (1998) Biochemistry 37,13250-13261; Milne et al., (1999) Biochemistry 38, 4768-4781). Together,the TOMM cyclodehydratase (C), dehydrogenase (B), and docking protein(D) comprise a functional, heterotrimeric thiazole/oxazole synthetase.

The genes encoding for this synthetase are typically located as adjacentopen reading frames in bacterial genomes, making such biosyntheticclusters relatively easy to identify using routine bioinformatic methods(Lee et al., (2008) Proc Natl Acad Sci USA 105, 5879-5884). TOMMbiosynthetic clusters often contain ancillary tailoring enzymes thatincrease the chemical complexity of this natural product family.

Although the unification of the TOMM family of natural products has onlyrecently emerged, the molecular structure and biological function ofsome TOMMs have long been established. Examples include microcin B17(DNA gyrase inhibitor), the cyanobactins (eukaryotic cytotoxins),streptolysin S (virulence-promoting cytolysin), and the thiopeptides(ribosome inhibitors) (Melby et al., (2011) Curr Opin Chem. Biol.,15(3):369-78). Bacillus amyloliquefaciens FZB42 is known to produce aplethora of complex small molecules, including bacillaene, difficidin,macrolactin, surfactin, fengycin, bacillomycin D, and bacillibactin(Chen et al., (2007) Nat Biotechnol 25, 1007-1014; Chen et al., (2006) JBacteriol 188, 4024-4036; Borriss et al., (2010) Int J Syst EvolMicrobiol, 61(Pt 8):1786-801).

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to a plantazolicin-likecompound having the structure:

(X¹)—(X²)₅—(X³)₂—(X⁴)₅—(X⁵)_(n)

or a pharmaceutically acceptable salt or ester thereof, wherein X¹ is

R¹ and R² are each independently hydrogen or lower alkyl; each X² isindependently an azole; each X³ is independently a hydrophobic aminoacid; each X⁴ is independently an azole or azoline; and each X⁵ isindependently an amino acid, wherein n is 1 or 2.

Another aspect of the present invention is directed to a plantazolicincompound having the structure

or a pharmaceutically acceptable salt or ester thereof.

It is still another aspect of the present invention to provide apharmaceutical composition comprising a plantazolicin-like compounddescribed herein or plantazolicin, and a pharmaceutically acceptablecarrier.

Yet another aspect of the present invention is a pharmaceuticalcomposition for treating or preventing a Bacillus anthracis infection ora Bacillus cereus infection wherein the therapy comprises administeringa pharmaceutical composition disclosed herein to an animal subject inneed thereof.

It is another aspect of the present invention to provide a method fortreating or preventing a Bacillus anthracis infection or a Bacilluscereus infection, the method comprising administering to apharmaceutical composition disclosed herein to an animal subject in needthereof.

Among other aspects of the present invention is a method for identifyinga plantazolicin-like protein by identifying a bacterial amino acidsequence exhibiting at least 50% amino acid identity to a plantazolicinprecursor peptide from Bacillus amyloliquefaciens FZB42; obtaining apost-translationally modified product of the bacterial amino acidsequence; and testing the post-translationally modified product of thebacterial amino acid sequence in a Bacillus anthracis growth inhibitoryassay, wherein ability to inhibit the growth of Bacillus anthracisindicates that the bacterial amino acid sequence encodes aplantazolicin-like protein.

Still other aspects of the present invention are directed to methods forproducing the plantazolicin. In one aspect, plantazolicin is produced bygrowing Bacillus amyloliquefaciens FZB42 cells in culture; collectingthe Bacillus amyloliquefaciens FZB42 cells, thereby obtaining theharvested Bacillus amyloliquefaciens FZB42 cells; obtaining a crudeplantazolicin extract from the harvested Bacillus amyloliquefaciensFZB42 cells; and purifying the plantazolicin compound from the crudeplantazolicin extract.

In another aspect, plantazolicin is produced by growing Bacillus pumiluscells in culture; collecting the Bacillus pumilus cells, therebyobtaining the harvested Bacillus pumilus cells; obtaining a crudeplantazolicin extract from the harvested Bacillus pumilus cells; andpurifying the plantazolicin compound from the crude plantazolicinextract.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts mass spectrometry-based structural elucidation of PZN. A,After biosynthetic processing, the final chemical structure of PZNfeatures N^(α),N^(α)-dimethylArg (green), two thiazoles (red), seven(methyl)oxazoles (blue), and one methyloxazoline (brown). The numberingscheme used for each original residue is given at the top of the figure.After treatment with mild acid, azoline heterocycles undergo hydrolyticring opening to the original amino acid (in this case, Thr).Stereochemical configuration is assumed to be identical to theribosomally produced peptide precursor. B, CID spectrum of PZN (m/z1336) acquired by LTQ-FT-MS. C, Same as B, except the parental ionanalyzed was hydrolyzed PZN (m/z 1354). *Denotes ions resulting from theloss of guanidine that localize the site of dimethylation to the α-amineof Arg. Localization to Arg is further supported by loss ofN^(α),N^(α)-dimethylArg (m/z 1180). **Denotes ions indicating that thesole azoline moiety of PZN is derived from the most C-terminal Thrresidue. All masses are given in Daltons (Da) and represent thesingly-charged ion. For proposed structures of the daughter ions, seeFIGS. 7-8.

FIG. 2 shows the effect of oxygenation during fermentation on theproduction of PZN. Cultivation at both high (biofermentor) and low(flasks) oxygen levels (see methods in Example 2) were grown for 24hours at 37° C. All samples were extracted and subjected tochromatography using an identical procedure. In all panels, verticallines were drawn at 14.7, 19.9, and 20.5 min. A, UV chromatogram (Abs266 nm) of FZB42 strain RSpMarA2 extract from high and low oxygenfermentation.

B, Same as A except the trace is the total ion chromatogram (TIC). C,Extracted ion chromatogram (EIC) of m/z 1336, 1338, and 1354 from a lowoxygen fermentation.D, Same as C except under high oxygenation conditions.

FIG. 3 shows an assessment of PZN antibiotic activity. A, The minimuminhibitory concentration (MIC) of HPLC-purified PZN was measured againsta panel of Gram-positive human pathogens. Values reported were theconcentration of PZN that inhibited 99% of the bacteria growth in amicrobroth dilution bioassay. *Due to separation difficulties,dihydroPZN was supplied as a 1:2:2 mixture of non-, mono-, anddihydrolyzed species (m/z 1338, 1356, 1374). B, PZN activity in an agardisk diffusion bioassay against B. anthracis Sterne. Upper left disk, 8μg kanamycin control (positive); upper right, solvent control(negative); lower disk, 100 μg PZN (200 μg gave a similar inhibitiondiameter). C, Visual appearance of live B. anthracis Sterne treated witha solvent control by DIC microscopy. D, Same as panel C, except cellswere treated with 4 μg/mL PZN. Scale bar is the same for panel C.

FIG. 4 depicts the PZN biosynthetic gene clusters. A, Open-reading framediagram showing the genetic organization of PZN clusters, which form asubclass of thiazole/oxazole-modified microcins (TOMMs). Genedesignations and predicted functions are color coded in the providedlegend. B, PZN precursor peptide (PznA) alignment. Shown in purple areconserved residues within the N-terminal leader region. *Denotes thePznA leader peptide cleavage site, which is known for BamA and BpumA butpredicted for the others. Color-coding indicates the posttranslationalmodification found at each residue in the BamA core region (otherprecursor peptide modifications are extrapolated from the knownstructure of PZN from FZB42). The conserved Arg (green) undergoes twomethylation events to yield N^(α),N^(α)-dimethylArg. Cys (red) areconverted to thiazoles while all blue residues become (methyl)oxazoles.The most C-terminal cyclizable position in BamA (brown, Thr) is left asan azoline heterocycle. Abbrev.: Bam, Bacillus amyloliquefaciens FZB42;Bpum, Bacillus pumilus ATCC 7061; Cms, Clavibacter michiganensis subsp.sepedonicus; Cur, Corynebacterium urealyticum DSM 7109; Blin,Brevibacterium linens BL2. Bam and Bpum are Firmicutes (Gram-positive,low % GC genome), while the other three species are Actinobacteria(Gram-positive, high % GC genome).

FIG. 5 shows the FTICR-MS of PZN (m/z 1336, broadband and CID spectrumof 2+ charge state). A, Broadband spectrum of HPLC-purified PZN on alinear ion trap MS (11 Tesla LTQ-FT). Visible are the singly and doublycharged positive ions of PZN. Due to the high mass accuracy of FT-MS (<5ppm error) and the known sequence of the precursor peptide (1, 2), themolecular formula of PZN was deduced from this mass measurement. The PZNmolecular formula (neutral species) is C₆₃H₆₉N₁₇O₁₃S₂ (monoisotopicmass, 1335.4702; error, 0.15 ppm). This formula required that 9 out ofthe 10 heterocyclizable residues were converted to the azole heterocycleand the remaining residue was left at the azoline oxidation state. Also,this formula required two methylation events (consistent with earlierdeletion studies) and leader peptide cleavage after Ala-Ala (see FIG. 4b). B, Collision induced dissociation (CID) spectrum of m/z 668.7(PZN²⁺). The fragmentation pattern of PZN in the doubly charged state ismarkedly different than that of the singly charged species shown in FIG.1B. The amino acid sequence for the PZN precursor peptide from B.amyloliquefaciens FZB42 (BamA) is color-coded by posttranslationalmodification as follows: N^(α),N^(α)-dimethylarginine (green), thiazoles(red), methyloxazoles and oxazoles (blue), and methyloxazoline (brown).Identified fragment ions are also plotted onto the BamA precursorsequence. The most diagnostic peaks for localizing posttranslationalmodifications resulted from Ile-Ile cleavage (green and brown masspeaks). These ions demonstrate that both methylation events are on theN-terminal fragment and that the sole azoline moiety is on theC-terminal fragment. *Fragment ions with the azoline as the mostC-terminal moiety spontaneously decompose, supporting the assignment ofthe C-terminal Thr as being converted to methyloxazoline in PZN(assigned in FIG. 7). Under the CID conditions employed, most peptidesfragment at the amide bond. The first step in TOMM biosynthesis,cyclodehydration, removes an amide bond from the peptide backbone.**Contiguous heterocycles thus preclude the formation of a completeseries of y⁺ and b⁺ ions and result in a CID spectrum that isfeatureless from m/z ˜710-1100. One non-amide cleavage is noted betweenarginine and cysteine (highest mass ion in the spectrum), which permitsthe methyl groups to both be localized to arginine. ̂Internal fragments(assigned in FIG. 7).

FIG. 6 depicts the UV-Vis spectrum of HPLC-purified PZN in DMSO acquiredon a Nanodrop 2000. The instrument was blanked on DMSO, which has a UVcut-off of approximately 245 nm. The extinction coefficient for PZN inDMSO is ε₂₆₀=560 M⁻¹ cm⁻¹. The λ_(max) in 80% acetonitrile/water is 266nm.

FIG. 7 shows the fragmentation map for PZN (m/z 1336). Fragmentationpathways in ion trap mass spectrometers are typically not sequential andmost often result from the product of a single cleavage event. Thus, thearrows in this diagram are for illustrative purposes and are not meantto represent an actual pathway. The most revealing masses are boxed, inaddition to the parent ion (PZN). The m/z 1277 structure (green text)results from the dissociation of guanidine from PZN and permits thelocalization of both methyl groups to the N-terminus of PZN. The m/z1145 structure (brown text) results from the loss of the C-terminal Pheresidue and CO. In conjunction with selective hydrolysis studies, m/z1145 and the subsequent azoline decomposition ions localize the soleazoline as the C-terminal Thr residue. Further, there are many examplesof neutral loss of acetaldehyde (C₂H₄O, exact mass=44.0262; not to beconfused with loss of carbon dioxide, exact mass=43.9898; >800 ppmdifference).

FIG. 8 shows the fragmentation map for hydrolyzed PZN (m/z 1354). Unliketheir aromatic azole counterparts, azoline heterocycles arehydrolytically unstable in mild acid and mild base. Selective acidichydrolysis of PZN was performed to convert the sole azoline heterocycleback to the original amino acid. This reinstated an amide bond that canbe located by subsequent MS^(n) analysis. As mentioned in FIG. 7, thearrows are for illustration purposes only and not meant to indicate thatthe ions fragment following the path shown. Ions that are duplicativewith those given in FIG. 7 are not replicated here. There were no casesof neutral loss of acetaldehyde following methyloxazoline hydrolysis tothreonine. This implies that loss of acetaldehyde (formation of azirine)is specific to methyloxazolines under the CID conditions that wereemployed. The loss of C₂H₄O was possible from hydrolyzed(Thr-containing) PZN, but only via dehydration (could also be α/β, drawnas (β/γ) and subsequent loss of acetylene. Other fragment ions ofinterest in this map confirmed the site of dimethylation to be theN-terminal amine.

FIG. 9 shows the CID spectra for deguanidinated PZN (m/z 1277) anddeguanidinated hydrolyzed PZN (m/z 1295). MS³ collision induceddissociation (CID) spectra for A, deguanidinated PZN (m/z 1277) and B,deguanidinated hydrolyzed PZN (m/z 1295). **Indicates loss ofacetaldehyde (44.0262 Da) from methyloxazoline (1277−44=1233;1194−44=1150). Note that this is only possible in panel A, where anintact heterocycle is found. The ions at m/z 575, 1150, and 1194demonstrate that the Arg was dimethylated on the amino terminus.Structural assignments are given for the fragments of deguanidinated PZNand deguanidinated hydrolyzed PZN in FIGS. 7 and 8, respectively.

FIG. 10 depicts the N-terminal labeling of PZN and desmethylPZN usingNHS-biotin. MALDI-TOF-MS results of NHS-biotin labeling for A, PZN (m/z1336) and hydrolyzed PZN (m/z 1354) and B, desmethylPZN (m/z 1308) andhydrolyzed desmethylPZN (m/z 1326). Abbreviation: desmethylPZN, dmPZN.Red traces are samples that included the NHS-biotin reagent while blacktraces are from control reactions that lacked NHS-biotin. Labeling wasonly observed with desmethylPZN, as indicated by the new species at m/z1534 and 1552. Addition of biotin gives a net mass increase of 226 Da(C₁₀H₁₄N₂O₂S). Specific labeling reactions are given in the methodssection.

FIG. 11 shows the ¹H-¹H-gCOSY of PZN. Assigned correlations are drawn onthe structure of PZN as thickened bonds. The brown circles indicatecorrelations deriving from the methyloxazoline protons (shown as brownbonds in structure). The red asterisk indicates that in the1D-¹H-spectrum, the signal from water was suppressed. This signal wasnot suppressed for the 2D experiment.

FIG. 12 shows the ¹H-¹H-TOCSY of PZN. Assigned correlations are drawn onthe structure of PZN as thickened bonds. The brown circles indicatecorrelations deriving from the methyloxazoline protons (shown as brownbonds in structure). The red asterisks on the 1D spectra indicate thesignal from water suppression. This signal was also suppressed for the2D experiment.

FIG. 13 depicts the ¹H-¹³C-gHMBC of PZN. Assigned correlations are drawnon the structure of PZN as red arrows. The green arrows/circles indicatecorrelations that localize the posttranslational methyl groups to theN-terminus. The brown arrows/circles indicate correlations thatdemonstrate the azoline is methyloxazoline.

FIG. 14 shows the predicted isotope pattern for PZN (m/z 1336). Theaverage mass is slightly heavier than the first isotope mass. Thisfigure was generated using iMass version 1.1 (freeware written by UrsRoethlisberger).

FIG. 15 shows the effect of oxygen levels during fermentation on theproduction of PZN. ESI-MS at selected time points from LCMS analysis(UV, TIC, EIC) is shown in FIG. 2. A, Under low oxygen conditions, PZN(m/z 1336) is the only species present in the 19.9 min elution. B, Underan oxygen saturated fermentation, PZN is found in the 20.5 min elution.C, As expected from the EIC's shown in FIG. 2, high oxygen fermentationyields an additional compound eluting at 14.7 min consistent withdihydroPZN (dhPZN, m/z 1338). The earlier elution of dhPZN relative toPZN is in agreement with azolines being more hydrophilic and basic thanazoles (azoles are not protonated with 0.1% formic acid). Right insetsfor all panels show a zoomed-in spectrum to highlight the isotopicpattern of the singly charged PZN species.

FIG. 16 depicts the localization of second azoline heterocycle ondihydroPZN (dhPZN). A, CID spectrum of dhPZN (m/z 1338) acquired usingLTQ-FT-MS. The heavier fragment ions are identical to those shown inFIG. 1, with the exception of each fragment being 2 Da heavier. The graybox depicts a zoomed-in region shown in panel B. Brown boxes highlighttwo ions demonstrating that an azoline heterocycle exists on each sideof the Ile-Ile. The location of the C-terminal azoline was localized tothe most C-terminal Thr. The location of the N-terminal azoline islikely to be the Thr adjacent to Ile due to similar sterics/electronics.However, the precise position cannot be concluded from this spectrum. B,Zoomed-in region from panel A (gray box). Diagnostic ions are boxed ingray and their respective (predicted) structures are drawn in the rightmargin.

FIG. 17 depicts the effect of oxygen levels during fermentation on theproduction of desmethylPZN: UV, TIC, and EIC traces of desmethylPZN (m/z1308), dihydrodesmethylPZN (m/z 1310), and hydrolyzed desmethylPZN (m/z1326). In each case presented, the low oxygen samples were prepared byshake flask fermentation of B. amyloliquefaciens strain RS33 (pznLdeletion, desmethylPZN producer) in 2 L of LB in 6 L flasks. High oxygensamples were prepared using a biofermentor with 5 L/min air input. Bothcultures were grown for 24 h at 37° C. All samples were extracted in anidentical fashion and subjected to identical chromatographic procedures(analytical C₁₈—HPLC) as described in the methods. In all panels,vertical lines are drawn at 14, 17, and 21 min. A, UV chromatogram (Abs272 nm) of RS33 extract from high and low oxygen fermentation. Thistrace shows that more chromophores absorbing light at 272 nm areproduced under high oxygen conditions. B, Same as A except the trace isthe total ion chromatogram (TIC). C, Extracted ion chromatogram (EIC) ofm/z 1308, 1310, and 1326 from low oxygen fermentation. Under theseconditions, the majority species is desmethylPZN (1308) with traceamounts of hydrolyzed desmethylPZN (1326). The 1310 trace that appearsto “coelute” with 1308 at 17 min is actually the second isotope peak of1308, not dihydrodesmethylPZN (see FIG. 14). D, Same as C except underhigh oxygenation conditions. The peaks at 14 and 16 min containprimarily dihydrodesmethylPZN (m/z 1310) while the peaks at 17 and 21min contain primarily desmethylPZN (m/z 1308). The species eluting at 14and 16 min are suspected to be regioisomers, as are the species elutingat 17 and 21 min. ESI-MS data at these selected time points are shown inFIG. 18.

FIG. 18 shows the effect of oxygen levels during fermentation on theproduction of desmethylPZN. ESI-MS at selected time points from LCMSanalysis (UV, TIC, EIC) is shown in FIG. 17. A, Under low oxygenconditions, hydrolyzed desmethylPZN (m/z 1326) is visible in the 14 minelution. B, As expected from the EIC's shown in FIG. 17, the 14 minelution is dominated by dihydrodesmethylPZN (m/z 1310) at the 14 minelution. C, Low oxygen fermentation and an elution of 17 min yieldsexclusively desmethylPZN (m/z 1308). As indicated by the ion purity andsignal to noise ratio in this spectrum, relative to the other panels,desmethylPZN was a majority product and easily separated under theconditions employed. D, Same as C but high oxygen conditions led to theproduction of a mixture of desmethylPZN and dihydrodesmethylPZN (ratio˜60:40, respectively). E, At 16 min under high oxygen conditions, 1310is the majority species produced, consistent with azolines being morehydrophilic than azoles. Right insets for panels C-E show a zoomed-inspectrum to highlight the isotopic pattern of the singly chargeddesmethylPZN species. The ratio of intensities given in FIG. 14 applies.

FIG. 19 shows the similarity/identity matrix of related (PZN-producing)biosynthetic proteins. Shown in yellow are amino acid identity scoresobtained by pairwise alignment using ClustalW2, which includes thestandard parameters for gap penalties. In blue are the correspondingamino acid percent similarity values, obtained by recording the ratio ofsimilar amino acids to the full protein sequence after alignment (no gappenalties). Abbreviations: PznJ, required biosynthetic protein ofunknown function; PznC, cyclodehydratase; PznD, docking protein; PznB,FMN-dependent dehydrogenase; PznE, suspected leader peptidase; and PznL,SAM-dependent methyltransferase. Abbreviations used are derived from thegenus and species name for each organism. Bam, Bacillusamyloliquefaciens FZB42; Bpum, Bacillus pumilus ATCC 7061; Cms,Clavibacter michiganensis subsp. sepedonicus; Cur, Corynebacteriumurealyticum DSM 7109; and Blin, Brevibacterium linens BL2. Bam and Bpumare Firmicutes, while the other three species are Actinobacteria.

FIG. 20 depicts the PZN production from Bacillus pumilus ATCC 7061.Cells were grown in an identical fashion to B. amyloliquefaciens. A, Thecell surface metabolites were extracted with methanol, dried,concentrated, and separated on a preparative C₁₈—HPLC column with UVmonitoring at 266 nm (λ_(max) for PZN). B, The 22-min (top), 23-min(middle), and 24-min (bottom) fractions from HPLC purification wereconcentrated and spotted on to the MALDI target with sinapic acid. Inthe earliest fraction, m/z 1354 (hydrolyzed PZN) is visible. In thelatter two fractions, m/z 1336 (PZN) is readily identified and pooledfor further analysis. C, HPLC purified PZN from B. pumilus was subjectedto high-resolution MS (LTQ-FT-MS), which verified the molecular formulato be consistent with PZN within the mass accuracy of the instrument (<5ppm). D, CID spectrum obtained upon isolation of the singly charged (m/z1336) precursor ion. This data is analogous to FIG. 1B (PZN from B.amylo. RSpMarA2). E, CID spectrum obtained upon isolation of the doublycharged (m/z 668) precursor ion. This data is analogous to FIG. 5B (PZNfrom B. amylo. RSpMarA2). Different instrumental settings had to beemployed to visualize the PZN ions, which were less abundant than fromthe B. amylo. overproducer (RSpMarA2) and required summing over manyscans. An unidentified contaminant and instrumental noise account forthe ions between m/z 750-1100.

FIG. 21 depicts the plantazolicin gene cluster. A, FZB42 PZN genecluster (9892 bp) and amino acid sequence of the precursor peptide. (−)marks a putative leader peptide processing site. B, Proposed function ofindividual PZN genes. Upon deletion of pznF and pznl, cpd1336 (PZN) wasdetected by mass spectrometry. Deletion of pznL resulted in desmethylPZN (m/z=1308 Da) while individual inactivation of all other testedgenes (pznABCJ) did not produce PZN.

Corresponding reference characters indicate corresponding partsthroughout the drawings.

DEFINITIONS AND ABBREVIATIONS

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor or RNA (e.g., tRNA, siRNA, rRNA, etc.). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction, etc.) ofthe full-length or fragment are retained. The term also encompasses thecoding region of a structural gene and the sequences located adjacent tothe coding region on both the 5′ and 3′ ends, such that the genecorresponds to the length of the full-length mRNA. The sequences thatare located 5′ of the coding region and which are present on the mRNAare referred to as 5′ untranslated sequences. The sequences that arelocated 3′ or downstream of the coding region and that are present onthe mRNA are referred to as 3′ untranslated sequences. The term “gene”encompasses both cDNA and genomic forms of a gene. A genomic form orclone of a gene contains the coding region, which may be interruptedwith non-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are removed or “spliced out” from thenuclear or primary transcript, and are therefore absent in the messengerRNA (mRNA) transcript. The mRNA functions during translation to specifythe sequence or order of amino acids in a nascent polypeptide.

The term “expression cassette” is used to define a nucleotide sequencecontaining regulatory elements operably linked to a coding sequence thatresult in the transcription and translation of the coding sequence in acell.

The term “plasmid” as used herein, refers to an independentlyreplicating piece of DNA. It is typically circular and double-stranded.

As used herein, Bacillus anthracis spore (or anthrax spore) is a smallreproductive body produced by B. anthracis bacteria. Such spores do notform normally during active growth and cell division. Rather, theirdifferentiation begins when a population of vegetative cells passes outof the exponential phase of growth, usually as a result of nutrientdepletion.

“Preventing” a disease refers to inhibiting the full development of adisease, for example preventing development of anthrax disease.Prevention of a disease does not require a total absence of infection.For example, a decrease of at least 50% can be sufficient.

“Treatment” or “treating” refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological condition,such a sign or symptom of anthrax disease (e.g., fever, ulcers, swollenlymph nodes, skin blisters). Treatment can also induce remission or cureof a condition, such as anthrax disease, including inhalational anthrax,gastrointestinal anthrax, oropharyngeal anthrax and cutaneous anthrax.

The term “pharmaceutically acceptable salt” refers to the relativelynon-toxic, inorganic and organic acid addition salts of compounds of thepresent invention. These salts can be prepared in situ in theadministration vehicle or the dosage form manufacturing process, or byreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thus formedduring subsequent purification. Representative salts include thehydrobromide, hydrochloride, sulfate, bisulfate, malate, citrate,flurbiprofen, ketoprofen, loxoprofen, diclofenac, etodolac,indomethacin, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, laurylsulphonate salts and the like. (See,for example, Berge et al. (1977) “Pharmaceutical Salts,” J. Pharm. Sci.66: 1-19).

“PZN” as used herein is an abbreviation for plantazolicin.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the comparison window, and multiplying theresult by 100 to yield the percentage of sequence identity.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443,by the search for similarity method of Pearson and Lipman (1988) Proc.Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Ausubelet al., Current Protocols in Molecular Biology (1995 supplement)).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95%identity over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecomplement of a test sequence. Optionally, the identity exists over aregion that is at least about 50 nucleotides in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotidesin length.

The term “similarity,” or percent “similarity,” in the context of two ormore polypeptide sequences, refer to two or more sequences orsubsequences that have a specified percentage of amino acid residuesthat are either the same or similar as defined in the 8 conservativeamino acid substitutions defined above (i.e., 60%, optionally 65%, 70%,75%, 80%, 85%, 90%, or 95% similar over a specified region), whencompared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Such sequences are then said to be “substantially similar.”Optionally, this identity exists over a region that is at least about 50amino acids in length, or more preferably over a region that is at leastabout 100 to 500 or 1000 or more amino acids in length.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to novel plantazolicin-like compounds,which are highly discriminating antibiotics (i.e., they arenarrow-spectrum antibiotics in that they are active against a selectedgroup of bacterial types and used for the specific infections arisingfrom these bacterial types). The plantazolicin-like compoundsstructurally belong to a family of thiazole/oxazole-modified microcins(TOMMs).

One aspect of the present invention is directed to a plantazolicin-likecompound having the structure: (X¹)—(X²)₅—(X³)₂—(X⁴)₅—(X⁵)_(n) or apharmaceutically acceptable salt or ester thereof, wherein X¹ is

R¹ and R² are each independently hydrogen or lower alkyl; each X² isindependently an azole; each X³ is independently a hydrophobic aminoacid; each X⁴ is independently an azole or azoline; and each X⁵ isindependently an amino acid, wherein n is 1 or 2.

In some embodiments, X² is selected from the group consisting of:pyrazole, imidazole, thiazole, oxazole, isoxazole, isothiazole, pyrrole,triazole, tetrazole, and pentazole, and is preferably thiazole oroxazole.

In some instances, X³ is selected from the group consisting of alanine(Ala), valine (Val), isoleucine (Ile), leucine (Leu), methionine (Met),phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp). Preferably,X³ is isoleucine, phenylalanine, or tryptophan.

In some embodiments, X⁴ is an azole selected from the group consistingof pyrazole, imidazole, thiazole, oxazole, isoxazole, isothiazole,pyrrole, triazole, tetrazole, and pentazole, or an azoline selected fromthe group consisting of pyrazoline, imidazoline, thiazoline, oxazoline,isoxazoline, isothiazoline, pyrroline, triazoline, tetrazoline, andpentazoline. X⁴ is preferably is thizole, oxazole or oxazoline.

In some instances, X⁵ is selected from the group consisting ofphenylalanine, tyrosine and tryptophan. In an exemplary preferredembodiment, X⁵ is phenylalanine.

Preferably, n is 1.

Another aspect of the present invention is directed to plantazolicinhaving the structure

or a pharmaceutically acceptable salt or ester thereof.

The plantazolicin-like compound described above can be synthesized byany methods known in the art, such as by total chemical synthesis,semi-synthesis or bacterial strain bioengineering.

In one embodiment, plantazolicin is isolated from Bacillusamyloliquefaciens FZB42. Bacillus amyloliquefaciens FZB42 is agram-positive, plant-growth promoting bacterium with a large capacity toproduce secondary metabolites with antimicrobial activity.

Plantazolicin, belonging to a class of TOMM molecules is produced from asmall precursor peptide that is posttranslationally modified to containthiazole and (methyl)oxazole heterocycles. These rings are derived fromCys and Ser/Thr through the action of a trimeric ‘BCD’ synthetasecomplex, which consists of a cyclodehydratase (C), dehydrogenase (B),and a docking protein (D).

In general, during TOMM biosynthesis, the precursor peptide is bound bythe BCD synthetase complex through specific motifs within the N-terminalleader sequence. After substrate recognition, heterocycles aresynthesized on the C-terminal core peptide over two enzymatic steps. Thefirst is carried out by a cyclodehydratase, which converts Cys andSer/Thr residues into the corresponding thiazolines and(methyl)oxazolines. A dehydrogenase then oxidizes the ‘azoline’ rings toyield ‘azole’ rings [thiazoles and (methyl)oxazoles], resulting in a netloss of 20 Da. The completion of TOMM biosynthesis includes theincorporation of ancillary modifications (e.g. dehydrations,methylations, macrocyclization, etc.), and leader peptide proteolysis.In many cases, the fully mature TOMM natural product is then activelyexported from the cell through the use of an ABC transport system.

The PZN biosynthetic 12-gene cluster spans nearly 10 kb of the FZB42chromosome as shown in FIG. 21. Furthermore, as shown in the examples,plantazolicin biosynthetic genes are transcribed into two polycistronicmRNAs (pznFKGHI and pznJCDBEL) and a monocistronic mRNA for pznA. Thegene coding for a precursor peptide was identified by a manual ORFsearch and found to be encoded between pznI (RBAM_(—)007440) and pznJ(RBAM_(—)007450) in the opposite direction. Although it is notannotated, this ORF (pznA) bears a robust Shine-Dalgarno sequence,AGGAGG, which lies 8 base pairs upstream of an AUG start codon. TheC-terminal region, also known as the core peptide was found to be richin residues that can be enzymatically cyclized to thiazoles and(methyl)oxazoles (2 Cys, 4 Thr, and 4 Ser).

The first operon (pznFKGHI) consists of genes predicted to be involvedin immunity, regulation, and transport (FIG. 21). The product of pznK(RBAM_(—)007410) is related to homodimeric repressor proteins of theArsR family. While not being bound to a particular theory, this proteinpossibly regulates the expression of other PZN genes through anunexplored mechanism. The second operon (pznJCDBEL) harbors the genesencoding for the enzymes responsible for converting the inactive PznAprecursor peptide into the mature, bioactive natural product. A summaryof the putative function of the members of the PZN gene cluster in B.amyloliquefaciens FZB42 is given in FIG. 21.

Based on sequence alignment with other known TOMM biosynthetic clustergenes, PznC is related to the TOMM cyclodehydratase and believed to actas one. PznD is highly similar to SagD from the SLS biosynthetic clusterand is termed the docking scaffold protein, while PznE is believed to bea leader peptidase.

In one embodiment, plantazolicin is produced from a precursor peptidehaving the amino acid sequence MTQIKVPTALIASVHGEGQHLFEPMAARCTCTTIISSSSTF(SEQ ID NO: 1). In another embodiment, the core region of the precursorpeptide has the sequence of RCTCTTIISSSSTF (SEQ ID NO: 2).

In another embodiment, the present invention is directed to a method forproducing plantazolicin by growing Bacillus amyloliquefaciens FZB42cells in culture; collecting the Bacillus amyloliquefaciens FZB42 cells,thereby obtaining the harvested Bacillus amyloliquefaciens FZB42 cells;obtaining a crude plantazolicin extract from the harvested Bacillusamyloliquefaciens FZB42 cells; and purifying plantazolicin from thecrude plantazolicin extract. In other embodiments, any other Bacillusamyloliquefaciens strain, whether mutated or not that is capable ofproducing PZN can be used in this method.

Using a protein BLAST search, it was discovered that thiazole/oxazolesynthetase proteins (PznBCD) from B. pumilus (protein IDs: EDW22765.1,EDW22903.1, and EDW23125.1, respectively) demonstrated a significantdegree of amino acid identity to those from FZB42 (PznB, 77%; PznC, 63%,and PznD, 82%). Moreover, the PZN genes from B. pumilus were found to beclustered and in identical order to that found in FZB42. Similarly toFZB42, B. pumilus is a plant saprophyte that produces an array ofantibacterial and antifungal natural products. Furthermore, the PznAcore peptide sequences from FZB42 and B. pumilus (unmarked, locatedbetween EDW23486.1 and EDW22932.1) were found to be 100% identical, andplantazolicin isolated from B. pumilus was identical to FZB42-isolatedPZN. Hence, it is another embodiment of the present invention to provideplantazolicin isolated from Bacillus pumilus.

Accordingly, it is another embodiment of the present invention toprovide a method for producing plantazolicin, the method comprisinggrowing Bacillus pumilus cells in culture; collecting the Bacilluspumilus cells, thereby obtaining the harvested Bacillus pumilus cells;obtaining a crude plantazolicin extract from the harvested Bacilluspumilus cells; and purifying plantazolicin from the crude plantazolicinextract.

In some embodiments related to the methods for producing PZN, cells,either B. amyloliquefaciens or B. pumilus, are grown in flasks. In otherembodiments, the cells are grown in biofermentors. This is especiallydesirable when larger quantities of plantazolicin are being produced.One of ordinary skill in the art can readily determine culture media andgrowth conditions necessary to produce plantazolicin in particularcells. Some of the exemplary conditions for both B. amyloliquefaciensand B. pumilus are shown in the examples.

The inventors also discovered that low and high oxygen levels usedduring cell growth, i.e. fermentation affected the production of PZN andits derivative metabolites. In particular, there was more PZN producedcompared to metabolites during low oxygenation, whereas high oxygenationresulted in greater production of metabolites, which were either lessactive (dihydroPZN) or inactive (desmethylPZN) compared to PZN. Hence,in some embodiments, the step of growing cells for production of PZN isperformed under low oxygen conditions. Low oxygenation refers toconditions such as regular growth of bacteria in flasks (i.e., at oxygenlevels present in air), whereas high oxygenation refers to oxygensupplementation, such as by supplying air at a rate of 5 L/min, the airbeing saturated in oxygen at a rate of approximately 1 L/min.

Cells are next harvested using any of the methods known in the art. Insome embodiments, cells are harvested by centrifugation or by any othermethod used in the art. One or more centrifugation steps can beperformed in order to obtain most of the cells. Following the harvestingstep, a crude PZN extract is obtained from the harvested cells. In someembodiments, the crude PZN extract is obtained by performing anon-lytic, methanolic extraction of the cellular surface of theharvested cells. Other solvents used for extraction can readily bedetermined by a skilled artisan. Exemplary conditions are shown in theexamples, and a skilled artisan can readily determine other conditionssuitable for crude PZN extraction.

A crude plantazolicin extract is next subjected to a purification step,which allows for separation of plantazolicin from other components inthe extract. In some embodiments of the present invention, plantazolicinis purified by high performance liquid chromatography (HPLC). Oneparticularly useful method of HPLC is reverse phase HPLC.

As shown in the examples and Table 3, plantazolicin was tested forgrowth inhibitory activity towards a wide range of bacteria. In total,18 strains from 16 distinct species were assayed for susceptibility toPZN (Table 3). It was determined that PZN exhibited activity primarilytowards Bacillus sp., including B. subtilis. PZN exhibited no activityagainst any tested Gram-negative organisms. To further define theselectivity within the Gram-positive organisms, the scope of PZNactivity towards a panel of ubiquitous human pathogens was evaluated,including methicillin-resistant Staphylococcus aureus (MRSA),vancomycin-resistant Enterococcus faecalis (VRE), Listeriamonocytogenes, Streptococcus pyogenes, and Bacillus anthracis strainSterne (a surrogate for the BSL3 pathogen, which lacks thepoly-D-glutamic acid capsule). Plantazolicin exhibited a potent growthinhibition of B. anthracis, whereas all other species were unaffected byPZN (with the exception of S. pyogenes, which was only inhibited by veryhigh concentrations of PZN). The action of PZN upon B. anthracis wasshown to be bactericidal, as described in the examples. Accordingly, aplantazolicin-like compound or plantazolicin can be used to inhibitgrowth of Bacillus species, which is useful for treating infectionscaused by Bacillus bacteria susceptible to PZN. By way of example andnot of limitation, a plantazolicin-like compound or plantazolicin can beused to treat B. cereus infection, which causes food poisoning inhumans. In particular, due to a potent bactericidal effect on B.anthracis, a plantazolicin-like compound or plantazolicin can be used asan effective, highly specific highly discriminating antibiotic againstanthrax infections.

Accordingly, it is an embodiment of the present invention to provide apharmaceutical composition comprising a plantazolicin-like compound,plantazolicin or a pharmaceutically acceptable salt or ester thereof anda pharmaceutically acceptable carrier. A plantazolicin-like compound,plantazolicin or a pharmaceutically acceptable salt or ester thereof canbe formulated as a pharmaceutical composition prior to administering toan animal subject, according to techniques known in the art.Pharmaceutical compositions of the present invention are characterizedas being at least sterile and pyrogen-free. Methods for preparingpharmaceutical compositions of the invention are within the skill in theart, for example as described in Remington's Pharmaceutical Science,17th ed., Mack Publishing Company, Easton, Pa., (1985).

The present pharmaceutical formulations comprise a plantazolicin-likecompound, plantazolicin or a pharmaceutically acceptable salt or esterthereof (e.g., 0.1 to 90% by weight) mixed with a pharmaceuticallyacceptable carrier. Suitable physiologically acceptable carriers arewater, buffered water, saline solutions (e.g., normal saline or balancedsaline solutions such as Hank's or Earle's balanced salt solutions),0.4% saline, 0.3% glycine, hyaluronic acid and the like.

The pharmaceutical composition of the present invention can beadministered orally, nasally, parenterally, intrasystemically,intraperitoneally, topically (as by drops or transdermal patch),bucally, sublingually or as an oral or nasal spray. In one preferredembodiment, the pharmaceutical composition of the present invention isadministered orally. In another preferred embodiment, the pharmaceuticalcomposition is given intravenously. In still another preferredembodiment, the pharmaceutical composition is given subcutaneously orintramuscularly.

A pharmaceutical composition of the present invention for parenteralinjection can comprise pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), carboxymethylcellulose and suitable mixturesthereof, vegetable oils (such as olive oil), and injectable organicesters such as ethyl oleate. Proper fluidity can be maintained, forexample, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

Injectable depot forms are made by forming microencapsulated matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

In some cases, to prolong the effect of the drugs, it is desirable toslow the absorption from subcutaneous or intramuscular injection. Thiscan be accomplished by the use of a liquid suspension of crystalline oramorphous material with poor water solubility. The rate of absorption ofthe drug then depends upon its rate of dissolution which, in turn, candepend upon crystal size and crystalline form. Alternatively, delayedabsorption of a parenterally administered drug form is accomplished bydissolving or suspending the drug in an oil vehicle. Prolongedabsorption of the injectable pharmaceutical form can be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

Solid dosage forms for oral administration include, but are not limitedto, capsules, tablets, pills, powders, and granules. In such soliddosage forms, the active compounds are mixed with at least onepharmaceutically acceptable excipient or carrier such as sodium citrateor dicalcium phosphate and/or a) fillers or extenders such as starches,lactose, sucrose, glucose, mannitol, and silicic acid, b) binders suchas, for example, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, and acacia, c) humectants such asglycerol, d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate, e) solution retarding agents such as paraffin, f) absorptionaccelerators such as quaternary ammonium compounds, g) wetting agentssuch as, for example, acetyl alcohol and glycerol monostearate, h)absorbents such as kaolin and bentonite clay, and i) lubricants such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof. In the case of capsules,tablets and pills, the dosage form can also comprise buffering agents.Solid compositions of a similar type can also be employed as fillers insoft and hard filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They can optionally contain opacifying agents. The compositions canalso release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes.

The pharmaceutical compositions of the present invention can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, solutions, suspensions,syrups and elixirs. In addition to the active compounds, the liquiddosage forms can contain inert diluents commonly used in the art suchas, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethyl formamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying agents, suspending agents,sweetening, flavoring, and perfuming agents.

Suspensions, in addition to a plantazolicin-like compound orplantazolicin, can contain suspending agents such as, for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminum metahydroxide, bentonite,agar-agar, and tragacanth, and mixtures thereof.

Alternatively, the composition can be pressurized and contain acompressed gas, such as nitrogen or a liquefied gas propellant. Theliquefied propellant medium and indeed the total composition arepreferably such that the active ingredients do not dissolve therein toany substantial extent. The pressurized composition can also contain asurface active agent. The surface active agent can be a liquid or solidnon-ionic surface active agent or can be a solid anionic surface activeagent. It is preferred to use the solid anionic surface active agent inthe form of a sodium salt.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (as for example calciumDTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodiumsalts (for example calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate).

One of ordinary skill in the art will appreciate that effective amountsof the agents of the invention can be determined empirically and can beemployed in pure form or, where such forms exist, in pharmaceuticallyacceptable salt, ester or prodrug form.

The pharmaceutical composition comprising a plantazolicin-like compoundor plantazolicin can be administered to a patient in order to preventand/or treat anthrax infection. It will be understood that, whenadministered to a human patient, the total daily usage of theplantazolicin compound or composition of the present invention will bedecided by the attending physician within the scope of sound medicaljudgment, and when administered to an animal, will be determined by aveterinarian. The specific therapeutically effective dose level for anyparticular patient will depend upon a variety of factors: the type anddegree of the cellular or physiological response to be achieved;activity of the plantazolicin compound; the age, body weight, generalhealth, sex and diet of the patient; the time of administration, routeof administration, and rate of excretion of the agent; the duration ofthe treatment; drugs used in combination or coincidental with thespecific agent; and like factors well known in the medical arts.

In some embodiments, a pharmaceutical composition comprising aplantazolicin compound is administered once daily. In other embodiments,plantazolicin is administered twice daily, and in still otherembodiments, it is administered three times a day. In some embodiments,a pharmaceutical composition comprises a plantazolicin compound in anamount from about 100 μg to about 100 mg. In other embodiments, thepharmaceutical composition comprises a plantazolicin compound in anamount from about 1 mg to about 50 mg.

As noted above, plantazolicin is bactericidal against B. anthracis, andas such pharmaceutical compositions described herein can be used totreat anthrax. Anthrax disease is caused by the bacterium Bacillusanthracis, a gram-positive, sporulating bacillus. B. anthracis is a soilbacterium and is distributed worldwide.

The disease can take on one of four forms: (1) cutaneous, the mostcommon, which results from contact with an infected animal or animalproducts; (2) inhalational, which is less common and a result of sporedeposition in the lungs, (3) gastrointestinal, and (4) oropharyngeal(back of the throat), both of which are due to ingestion of infectedmeat.

The cutaneous disease constitutes the majority (up to 95%) of anthraxcases. Anthrax usually develops in cattle, horses, sheep, and goats, andas such, is a major veterinary concern. Animals can contract the sporeswhile grazing. Humans can contract anthrax from inoculation of minorskin lesions with spores from infected animals, their hides, wool orother products, such as infected meat (Franz et al. (1997) J. Am. Med.Assoc. 278(5): 399-411). Anthrax in humans is rarer than in animalsunless the spores are spread intentionally.

Anthrax disease occurs when spores enter the body, germinate to thebacillary form, and multiply. In cutaneous disease, spores gain entrythrough cuts, abrasions, or in some cases through certain species ofbiting flies. Germination is thought to take place in macrophages, andtoxin release results in edema and tissue necrosis but little or nopurulence, probably because of inhibitory effects of the toxins onleukocytes. Generally, cutaneous disease remains localized, although ifuntreated it may become systemic in up to 20% of cases, withdissemination via the lymphatics. In the gastrointestinal form, B.anthracis is ingested in spore-contaminated meat, and may invadeanywhere in the gastrointestinal tract. Transport to mesenteric or otherregional lymph nodes and replication occur, resulting in dissemination,bacteremia, and a high mortality rate. Symptoms include nausea, loss ofappetite, vomiting, fever, abdominal pain, vomiting of blood and severediarrhea. Death results in 25%-60% of cases.

In cases of inhalation of anthrax spores, after deposition in the lowerrespiratory tract, spores are phagocytized by tissue macrophages andtransported to hilar and mediastinal lymph nodes. The spores germinateinto vegetative bacilli, producing a necrotizing hemorrhagicmediastinitis (Franz et al., supra). Symptoms include fever, malaise andfatigue, which can easily be confused with the flu. The disease mayprogress to an abrupt onset of severe respiratory distress with dyspnea,stridor, diaphoresis and cyanosis. Death usually follows within 24 to 36hours.

The average incubation period for anthrax is 1 to 7 days, but it cantake 60 days or longer for symptoms to develop. Symptoms depend on howthe infection was acquired. For humans, anthrax is a particularlyfearsome biological warfare agent, not only because of its deadliness,but also because anthrax weapons are relatively easy to produce anddeliver. Production of anthrax spores requires little more than basiclaboratory equipment and growth media. Anthrax weapons may be comprisedof an anthrax source and an industrial sprayer that can produce aerosolparticles of the appropriate size for victims to inhale. Such sprayers,for instance, can be mounted on low flying airplanes or other vehiclesand used to spread anthrax over a wide area. Because of the ease andrelatively small expense involved in producing and delivering anthraxweapons, such weapons are potentially highly attractive weapons of massdestruction for terrorist groups. Thus, in addition to potentialorganized military conflicts that may give rise to the use of suchweapons, terrorist organizations are a potential threat for the use ofsuch weapons in airports, office buildings and other centers of humanactivity.

Currently, B. anthracis infections are treated with variousbroad-spectrum antibiotics. In order to completely eliminate B.anthracis, antibiotic treatment often requires over 60 days ofadministration. Consequently, the current method of treatment increasesthe dangers of multi-drug resistance. Multi-drug resistance arises fromhorizontal gene transfer of drug-resistant bacteria and has lead to thegeneration of many harmful infectious diseases including, but notlimited to, Vancomycin-resistant enterococcus (VRE) andMethicillin-resistance Staphlococcus aureus (MRSA).

Most current treatments of bacterial infections kill off the humanintestinal bacteria which has two negative side effects: the “healthy”bacteria serve as a reservoir for antibiotic resistance and keep otherpathogens at bay. Prolonged, broad-spectrum antibiotics leave patientsat risk for secondary infections that are harder to treat that theprimary infection. A plantazolicin-like compound and plantazolicin, bothbeing highly discriminating antibiotics, provides numerous advantagesover the currently used antibiotics, such as high specificity and lowrisk of developing multi-drug resistance.

Accordingly, it is one embodiment of the present invention to provide amethod for treating or preventing anthrax in a patient by administeringto the patient any of the pharmaceutical compositions comprising aplantazolicin-like compound or plantazolicin that are described herein.In one embodiment, the patient is a human. In another embodiment, thepatient is an animal. The animal is preferably selected from a dog, cat,horse, sheep, goat, or cow. Any of the anthrax infections such ascutaneous, inhalation and gastrointestinal anthrax can be treated usinga plantazolicin-like compound or plantazolicin. In some embodiments, aplantazolicin-like compound or plantazolicin is used to treat or preventanthrax is administered orally, intravenously, subcutaneously orintramuscularly. In other embodiments, the daily dosage used to treatanthrax is from about 100 μg to about 100 mg of plantazolicin orplantazolicin-like compound, and in still other embodiments, the dailydosage of PZN or PZN-like compound is from about 1 mg to about 50 mg.

As noted above, B. pumilus produced PZN identical to the one produced byBacillus amyloliquefaciens FZB42. The search for other PZN-like proteinswas performed using a protein BLAST search where each PZN gene productwas used as the query sequence. B. pumilus ATCC 7061 was a top result inthe sequence search after FZB42, and the additional three PZN-likebiosynthetic clusters were found in the Actinobacteria phylum includingClavibacter michiganensis subsp. sepedonicus (potato pathogen),Corynebacterium urealyticum DSM 7109 (human skin-associated bacterium,causative agent of some urinary tract infections), and Brevibacteriumlinens BL2 (human skin-associated bacterium).

Another embodiment of the present invention is to provide a method foridentifying a plantazolicin-like protein, wherein the method comprisesidentifying a bacterial amino acid sequence exhibiting at least 50%amino acid identity to a plantazolicin precursor peptide from Bacillusamyloliquefaciens FZB42; obtaining a post-translationally modifiedproduct of the bacterial amino acid sequence; and testing thepost-translationally modified product of the bacterial amino acidsequence in a Bacillus anthracis growth inhibitory assay, wherein theability to inhibit the growth of Bacillus anthracis indicates that thebacterial amino acid encodes a plantazolicin-like protein.

The identification of a bacterial amino acid sequence exhibiting atleast 50% amino acid identity to the plantazolicin precursor peptide canbe used to search for a plantazolicin-like biosynthetic gene cluster ina bacterial genome. Alternatively, any other polypeptide from Bacillusamyloliquefaciens FZB42 can be used as a reference sequence to findother plantazolicin-like gene cluster products. By way of example andnot of limitation, PznE can be used to search for otherplantazolicin-like leader peptidases, which can then be used to searchfor plantazolicin-like biosynthetic clusters.

For sequence comparison, typically one sequence, such as plantazolicinin this case acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters can be used, or alternativeparameters can be designated. The sequence comparison algorithm thencalculates the percent sequence identities for the test sequencesrelative to the reference sequence, based on the program parameters.

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins and Sharp (1989) CABIOS 5:151-153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal. (1984) Nuc. Acids Res. 12:387-395).

Another example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The protein sequence search shows sequences in order of highest tolowest sequence identity to the reference sequence. Any bacterialsequences identified in the protein sequence search as exhibiting atleast 50% identity to the reference sequence can be further tested toconfirm whether they are indeed plantazolicin-like sequences. One way ofconfirming is to test a post-translationally modified product of thebacterial amino acid sequence in a Bacillus anthracis growth inhibitoryassay. In such an assay, the ability of the post-translationallymodified product of the bacterial amino acid sequence to inhibit thegrowth of Bacillus anthracis indicates that the bacterial amino acidencodes a plantazolicin-like protein.

In an exemplary embodiment, the post-translationally modified product ofthe bacterial amino acid sequence can be obtained by growing bacteriacontaining a gene for the bacterial amino acid sequence underconditions, which allow for transcription of such gene, and forpost-translational processing of a polypeptide encoded by the gene. Oneskilled in the art can determine such conditions without undueexperimentation. Some of the parameters that can be varied include mediacompositions, aeration conditions, incubation times and temperatures.

Molecular biological techniques, biochemical techniques, andmicroorganism techniques as used herein are well known in the art andcommonly used, and are described in, for example, Sambrook J. et al.(1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor andits 3rd Ed. (2001); Ausubel, F. M. (1987), Current Protocols inMolecular Biology, Greene Pub. Associates and Wiley-interscience;Ausubel, F. M. (1989), Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates and Wiley-interscience; Innis, M. A. (1990), PCRProtocols: A Guide to Methods and Applications, Academic Press; Ausubel,F. M. (1992), Short Protocols in Molecular Biology: A Compendium ofMethods from Current Protocols in Molecular Biology, Greene Pub.Associates; Ausubel, F. M. (1995), Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates; Innis, M. A. et al. (1995), PCR Strategies,Academic Press; Ausubel, F. M. (1999), Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Wiley, and annual updates; Sninsky, J. J. et al. (1999), PCRApplications: Protocols for Functional Genomics, Academic Press; and thelike. Relevant portions (or possibly the entirety) of each of thesepublications are herein incorporated by reference.

Having described the invention in detail, it will be apparent thatmodifications and variations are possible without departing from thescope of the invention defined in the appended claims.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present invention.

Example 1

Strain Construction.

The B. amyloliquefaciens strains and plasmids used in this study aresummarized in Table 1.

TABLE 1 Bacterial strains and plasmids used in this study StrainsDescription Source/reference Bacillus subtilis DSM10^(T) 168, trpC2,type strain DSMZ Braunschweig CU1065 168, trpC2 attSPβ Butcher et al.,Mol HB0042 168, trpC2 attSPβ sigW::kan Microbiol. 60: 765-82, 2006Bacillus megaterium 7A/1 Indicator strain for polyketides Laboratorystock Bacillus amyloliquefaciens FZB42 Wild type Idriss et al., 2002,Microbiology 148: 2097-109 CH5 FZB42 sfp::ermAM yczE::cm Chen, X. 2009.Whole genome analysis of the plant growth-promoting RhizobacteriaBacillus amyloliquefaciens FZB42 with focus on its secondarymetabolites. Dissertation. HU- Berlin. RSpMarA2 Insertion of pMarA inCH5: degU::kan Described herein RS6 sfp::ermAM, bac::cmR, deficient inlipopeptides, Chen et al., 2009, J polyketides and bacilysin Biotechnol.140: 38-44 RS26 (ΔpznB) RS6 ΔRBAM_007480::spc does not produce Describedherein PZN RS27 (ΔpznI) RS6 ΔRBAM_007440::spc produces PZN Describedherein RS28 (ΔpznJ) RS6 ΔRBAM_007450::spc does not produce Describedherein PZN RS29 (ΔpznF) RS6 ΔRBAM_007400::spc produces PZN Describedherein RS31 (ΔpznC) RS6 ΔRBAM_007460::spc does not produce Describedherein PZN RS32 (ΔpznA) RS6 ΔpznA::spc does not produce PZN Describedherein RS33 (ΔpznL) RS6 ΔRBAM_007500::spc produces desmethyl- Describedherein PZN, 1308 Da Plasmids pGEM-T Ap^(r), lacZ{grave over ( )} PromegapMarA plasmid containing mariner transposon TnYLB-1 Le Breton et al.,2006, Appl Environ Microbiol 72: 327-33 pIC333 plasmid with spc cassetteT. Msadek, Institute Pasteur, Paris, France pRS26a pGEM-T with 2700 bppznB Described herein pRS26b pGEM-T with pznB::spc Described hereinpRS27 pGEM-T with SOE fusion-product Described herein RBAM_007440/spcpRS28 pGEM-T with SOE fusion-product Described herein RBAM_007450/spcpRS29 pGEM-T with SOE fusion-product Described herein RBAM_007400/spcpRS31a pGEM-T with 2600 bp pznC Described herein pRS31b pGEM-T withpznC::spc Described herein pRS32a pGEM-T with 2300 bp flanking regionpznA Described herein pRS32b pGEM-T with pznA::spc Described herein

Bacillus and indicator strains were cultivated routinely onLuria-Bertani broth (LB) medium solidified with 1.5% agar. Forproduction of PZN, a medium containing: 40 g soy peptone, 40 g dextrin10, 1.8 g KH₂PO₄, 4.5 g K₂HPO₄, 0.3 g MgSO₄×7H₂O, and 0.2 ml KellyTtrace metal solution per L was used. KellyT trace metal solution: 25 mgEDTA disodium salt dihydrate, 0.5 g ZnSO₄×7H₂O, 3.67 g CaCl₂×2H₂O, 1.25g MnCl₂×4H₂O, 0.25 g CoCl₂×6H₂O, 0.25 g ammonium molybdate, 2.5 gFeSO₄×7H₂O, 0.1 g CuSO₄×5H₂O; adjust to pH 6 with NaOH, 500 ml H₂O.

The media and buffers used for DNA transformation of Bacillus cells wereprepared according to Kunst and Rapoport (J. Bacteriol. 177:2403-2407,1995). Competent cells were prepared as previously described (Koumoutsiet al., 2004. J. Bacteriol. 186:1084-96). Mutants were obtained aftertransformation of the FZB42 derivatives with linearized, integrativeplasmids containing resistance cassettes flanked by DNA regionshomologous to the FZB42 chromosome. The oligonucleotides used for strainconstruction are listed in Table 2.

TABLE 2Oligonucleotides used for gene replacement and Slice Overlap Extension (SOE) PCR Oligonucleotide Sequence (5′ to 3′)Spectinomycin resistance cassette Spc-fwCTCAGTGGAACGAAAACTCACG (SEQ ID NO: 3) Spc-rvTAAGGTGGATACACATCTTGTC (SEQ ID NO: 4) pRS26a/b pznB-fwATCCATATCGCCAATCATACGG (SEQ ID NO: 5) pznB-rvGGAATCAATACCTGTCAGTTCG (SEQ ID NO: 6) pRS31a/b pznD-fwATTGACTAGGAGGTATTGGACG (SEQ ID NO: 7) pznD-rvTTCTATTGAATAGGAGGAGGCG (SEQ ID NO: 8) pRS32a/b 007400cst-fwTGGAATGCTCTTTCCGCAGTAC (SEQ ID NO: 9) 007400cst-rvGTAACTCTGTTTCCACGTAACC (SEQ ID NO: 10) SOE PCR 7400 rvTCTTCATCACGCAAATCAGTGC (SEQ ID NO: 11) 7400 fwCCGCATAAACGGGAATTGGAAG (SEQ ID NO: 12) Spc in 7410TCTATAGAAACTTCTCTCAATTAGAAAAGAAAAGGGCAAGGAAA TGAG (SEQ ID NO: 13)7410 in spc ACTCATTTCCTTGCCCTTTTCTTTTCTAATTGAGAGAAGTTTCTATAG (SEQ ID NO: 14) Start in spcCTTTGTAAAAAGAGGAGCCTGTCTTATGAGCAATTTGATTAACGG (SEQ ID NO: 15)Spc in start TTTTTCCGTTAATCAAATTGCTCATAAGACAGGCTCCTCTTTTTACAAAG (SEQ ID NO: 16) 7430 in spcGCTGGGACTAAAAGGAGAGCGGGAAATGAGCAATTTGATTAACG G (SEQ ID NO: 17)Spc in ORF2 TTCTATAGAAACTTCTCTCAATTAGATTTAATATAAAGAAGCATAGACC (SEQ ID NO: 18) Spc in 7430TTTTTCCGTTAATCAAATTGCTCATTTCCCGCTCTCCTTTTAGTCC CAGC (SEQ ID NO: 19)ORF2 in spc TGGTCTATGCTTCTTTATATTAAATCTAATTGAGAGAAGTTTCTATAG (SEQ ID NO: 20) 7440 rv TCACGTCCAATACCTCCTAGTC (SEQ ID NO: 21)7440 fw ATCGACAGAGGGCAGATTATCG (SEQ ID NO: 22) ORF2 in spc for 7450GATTATTGACTAGGAGGTATTGGACATGAGCAATTTGATTAACG fw G (SEQ ID NO: 23)7460 in spc for 7450  GTTTGTTGAGACATCTGTATTCCTCCCTAATTGAGAGAAGTTTCT rvATAG (SEQ ID NO: 24) 7450 rv TAATGTCGTCCATTTACTCACC (SEQ ID NO: 25)7450 fw TTGGCTCGAATAAATGTTGACC (SEQ ID NO: 26) Spc in ORF2 for 7450TTTTTCCGTTAATCAAATTGCTCATGTCCAATACCTCCTAGTCAAT rv AATC (SEQ ID NO: 27)Spc in 7460 for 7450  TTCTATAGAAACTTCTCTCAATTAGGGAGGAATACAGATGTCTCA fwACAAAC (SEQ ID NO: 28) End in spc for 7500 CGCTTAGACCCTAAAGATATACTTTCTCTAATTGAGAGAAGTTTC rv TATAG (SEQ ID NO: 29)7490 in spc for 7500  AACTCTTTGGAGGTGTCACAGTTATATGAGCAATTTGATTAACGG fw(SEQ ID NO: 30) 7500 fw AAGGTCCTAGACGCCCTATTCC (SEQ ID NO: 31) 7500 rvGATGTGTAGTTTTCAACGCTCG (SEQ ID NO: 32) Spc in end for 7500 CTATAGAAACTTCTCTCAATTAGAGAAAGTATATCTTTAGGGTCT fw AAGCG (SEQ ID NO: 33)Spc in 7490 for 7500  CCGTTAATCAAATTGCTCATATAACTGTGACACCTCCAAAGAGTT rvTACC (SEQ ID NO: 34) Primers for RT-PCR pznF (fw and rv)GGATTATTGCGTACTCCGTTTC (SEQ ID NO: 35)CTGCCTCCGCCAATAAATG (SEQ ID NO: 36) pznK (fw and rv)ATGCCAAAGTACGGTTGGG (SEQ ID NO: 37) CTCCTTGTAGGCTGCTTTCC (SEQ ID NO: 38)pznG (fw and rv) CCACAGGATATCAGCCTTGAAG (SEQ ID NO: 39)CGATAATCTGCCCTCTGTCG (SEQ ID NO: 40) pznH (fw and rv)CGCTCGCTCAAATTGAAACG (SEQ ID NO: 41)ACAACAACCCACAGATACGC (SEQ ID NO: 42) pznI (fw and rv)TAGCCTGGAAGCAGAGGGTA (SEQ ID NO: 43)ACTTTTGGCAGGTGACAACC (SEQ ID NO: 44) pznA (fw and rv)GGAGGAGGTAACAATTATGACTCAA (SEQ ID NO: 45)GGTACAGGTACAGCGTGCAG (SEQ ID NO: 46) pznJ (fw and rv)TTGGATATCGGAATCGAGTTG (SEQ ID NO: 47)CGGATGCCCAATTATCTGTT (SEQ ID NO: 48) pznC (fw and rv)TCATGTCCCTTGGTTGTGTG (SEQ ID NO: 49)GCCGTGATACCATACTTGAGG (SEQ ID NO: 50) pznD (fw and rv)CGCGATGTAGATGACGTTTG (SEQ ID NO: 51)GATTGGCGATATGGATTAGTTG (SEQ ID NO: 52) pznB (fw and rv)AAGGCATGCCACTAATTTGG (SEQ ID NO: 53)GATAAAGAGCTCCGCCAGAA (SEQ ID NO: 54) pznE (fw and rv)CATAGCAATAATGCGTACGGTG (SEQ ID NO: 55)GAGACATTGTCGGCGAAGA (SEQ ID NO: 56) pznL (fw and rv)GATGAGAGGGAAACCTCATCC (SEQ ID NO: 57)CTCCCAAACTGTTCCTGTCC (SEQ ID NO: 58)

Spectinomycin (90 μg/ml) was used for selecting transformants. Geneinterruption strains were obtained as follows: PznB RS26: A 2.7 kb PCRfragment was amplified from FZB42 chromosomal DNA using primers pznB-fwand pznB-rv. The fragment was then cloned into pGEM-T, yielding plasmidpRS26a. Plasmid pRS26b was obtained by insertion of a spectinomycinresistance cassette, which was subcloned by PCR using the spc-fw andspc-rv primers and the pIC333 plasmid as a template. The cassette wasplaced into the central region of the insert and digested with BglII andBamHI. PznC RS31: With primers pznC-fw and pznC-rv, a 2.6 kb fragmentcontaining pznC was amplified by PCR and cloned into vector pGEM-T-Easyyielding plasmid pRS31a. A central fragment of the insert was removed bydigestion with Eco105I and replaced with the spectinomycin resistancecassette, yielding pRS31b. PznA RS32: With primers 007400cst-fw and007400cst-rv, a 2.3 kb fragment encoding the unannotated precursorpeptide, pznA, was amplified by PCR and cloned into vector pGEM-T-Easy,yielding plasmid pRS32a. The precursor peptide gene was cleaved byBsp14071 and interrupted by insertion of a spectinomycin resistancecassette, yielding pRS32b.

The mutants RS27, RS28, RS29 and RS33 were generated by gene splicingusing the overlapping extension (SOE) method (Horton et al, 1990,Biotechniques 8:528-35). This method assists in avoiding possible polareffects caused by interrupted reading frames. SOE PCR fusion productswere generated using the primers listed in Table 2 and the spectinomycingene of pIC333. A-tailing of the Pfu-PCR product was performed accordingto the Promega pGEM-T protocol and ligated into pGEM-T yielding pRS27,pRS28 and pRS29. For mutant RS33, the PCR product was used directly fortransformation.

Mutant RSpMarA2 was isolated from a mariner-based (pMarA) transposonlibrary prepared in strain CH5 according to Breton et al. (Appl EnvironMicrobiol 72:327-33, 2006). In this transposon mutant, pMarA wasintegrated into the degU gene, which is a global transcriptionalregulator that activates the bacillomycin D promoter.

Bioassay.

LB-Agar (20 ml) was mixed with 0.5 ml of the indicator strain(OD₆₀₀˜1.0). 10 μl of purified PZN suspended in water was spotted on theagar and incubated for 16 h at 22° C. The growth suppression activity ofPZN was observed as clear zone.

Purification of Plantazolicin.

A cell surface extract from a 250 ml culture of strain RSpMarA2 wascollected using the previous method. During concentration under reducedpressure, plantazolicin precipitated. The precipitate was washed 3 timeswith deionized water, resulting in crude, desalted plantazolicin. Pureplantazolicin was obtained using RP-HPLC (Grom-Sil ODS-5 ST, 20×250 mm,Alltech-Grom, Rottenburg-Hailfingen) with a linear gradient elution of40-70% aqueous acetonitrile with 0.1% v/v formic acid over 40 min at aflow rate of 15 ml/min.

RT-PCR.

Total RNA was isolated with the Qiagen RNeasy Mini Kit. Cells (1.0OD₆₀₀) were harvested from M9 minimal media supplemented with BMEvitamin mix (Cat. No. B6891) and ATCC trace mineral solution (Cat. No.MD-TMS) and treated with the Qiagen RNAprotect Bacteria Reagent.Harvested cells were resuspended in 250 μl of 10 mM tris (pH 8.5) with15 mg/ml lysozyme and 5 μl proteinase K (20 mg/ml) and digested for 1 hat 22° C. with gentle agitation. A DNase I digestion was performed forpznE and pznL using the Qiagen RNase Free DNase set. DNase I (7 μl) andRDD DNA digest buffer (7 μl) were used to hydrolyze contaminating DNAfor 20 min at 22° C. The RNA isolation protocol was then followed tomanufacturer's instructions. To minimize background, a DNase I digestion(5 μl) was executed to the RNA samples and placed at 37° C. for 20 min.It should be noted that this was the second DNase I digest for pznE andpznL. Samples were column purified using the RNA cleanup protocol in theRNeasy Mini Handbook (Qiagen). Digestion and cleanup were repeated forall RNA samples, excluding those used to analyze pznE and pznF. cDNA wasprepared with commercially available RT-PCR kits using 1 μg of RNA andthe primers listed in Table 2.

Reverse Transcriptase-PCR.

Transcription of all 12 pzn genes in M9 minimal media was confirmed byRT-PCR. All amplicons migrated with their expected sizes (Table 2). Inaddition to confirming transcription, the intergenic regions of the PZNbiosynthetic cluster were assessed to determine if the mRNA waspolycistronic. Using the appropriate primers from adjacent genes, it wasdetermined that the biosynthetic genes were transcribed into twopolycistronic mRNAs (pznFKGHI and pznJCDBEL) and a monocistronic mRNAfor pznA. Amplification of the region between pznE and pznL resulted ina band that was visible only under extreme contrast (data not shown).

Plantazolicin was tested for its ability to inhibit the growth ofGram-positive bacteria. In the agar bioassays, where growth inhibitionis indicated by a clear zone, plantazolicin was shown to be growthinhibitory towards most of the gram-positive Bacilli surveyed,especially B. megaterium and B. subtilis HB0042 (Table 3).

TABLE 3 Activity spectrum of plantazolicin Indicator strain Result^(a)Source or reference^(b) Bacillus brevis ATCC8246 + ATCC Bacillussubtilis 168 + Peric-Concha and Long (2003) Drug Discov Today 8,1078-1084 Bacillus cereus ATCC14579 + ATCC Bacillus licheniformisATCC9789 + ATCC Micrococcus luteus + Laboratory collection Bacilluspumilus − Laboratory collection Bacillus subtilis CU1065 + Peric-Conchaand Long (2003) Drug Discov Today 8, 1078-1084 Bacillus subtilis HB0042++ Peric-Concha and Long (2003) Drug Discov Today 8, 1078-1084 Bacillussphaericus + Laboratory collection Paenibacillus polymyxa − Laboratorycollection Paenibacillus granivorans + Laboratory collection Bacillusmegaterium 7A1 ++ Laboratory collection Arthrobacter sp. − Laboratorycollection Staphylococcus aureus − Laboratory collection E. coli K12 −Laboratory collection Klebsiella terrigena − Laboratory collectionPseudomonas sp. − Laboratory collection Erwinia caratovora − Laboratorycollection ^(a)Degree of inhibition in a bioassay: ++: inhibition; +:weak inhibition; −: no inhibition ^(b)ATCC: American Type CultureCollection

Example 2

Production and Purification of PZN.

Overnight cultures (4×20 mL) of B. amyloliquefaciens FZB42 strainRSpMarA2 (Δsfp, yczE, degU) were used to inoculate 4×6 L flasks with 2 Lof Luria Burtani (LB) broth supplemented with chloramphenicol (7 μg/mL)and kanamycin (7 μg/mL). Cultures were grown with shaking for 48 h at37° C. Cells were harvested by centrifugation (4,000×g), washed withTris-buffered saline (pH 8.0), and harvested a second time. Crude PZNwas obtained by a non-lytic, methanolic extraction of the cellularsurface. Cells were resuspended in MeOH (10% culture volume) andanhydrous Na₂SO₄ (5 g/L culture). The cell mixture was agitated byvortex (45 s) and equilibrated for 15 min at 22° C. The supernatant wasretained after centrifugation (4,000×g), vacuum filtered with Whatmanfilter paper, and rotary evaporated to dryness to yield about 100 mg/Lof a yellowish-brown solid. This crude material was dissolved in 80%aqueous MeCN (10 mL for 8 L culture), where the sample separated intotwo layers. The top organic layer was retained and concentrated forinjection onto an Agilent 1200 series liquid chromatograph that wasfitted inline to an Agilent 6100 Series Quadrupole LC/MS. Forpreparative purposes, PZN was reverse phase purified using a ThermoBETASIL C18 column (250 mm×10 mm; pore size: 100 Å; particle size: 5 μm)at a flow rate of 4 mL/min. A gradient of 65-85% MeOH with 0.1% formicacid over 32 min was used. The fractions containing PZN (as monitored byA₂₆₆ and MS) were collected into 20 mL borosilicate vials and thesolvent removed in vacuo. The isolated yield for PZN following thisprocedure was routinely 150-200 μg/L culture. Mutant RS33 (Asfp, bac,pznL) was prepared similarly, with the only exceptions being a 24 hfermentation, substitution of spectinomycin (90 μg/mL) for kanamycin,and elimination of the TBS wash.

Production of PZN (Elevated Oxygen).

Increased aeration of B. amyloliquefaciens FZB42 strains RSpMarA2 andRS33 was achieved using a New Brunswick Scientific BioFlo 110 Fermentersystem. RSpMarA2 and RS33 (9 L) were cultured at 37° C. with 250 rpmstirring for 24 h. Air was supplied at 5 L/min (saturated in oxygen, ˜1L/min).

Determination of Minimum Inhibitory Concentration (MIC).

B. anthracis strain Sterne was grown to stationary phase in a 10 mL LBculture at 37° C. The culture was adjusted to OD₆₀₀ of 0.01 in LB brothand added to 96-well plates. 2-fold dilutions of PZN (5 mg/mL in 80%MeCN) were added to the cultures (0.5-128 μg/mL). Kanamycin was addedsimilarly to control samples, with dilutions from 1-32 μg/mL. Coveredplates were incubated at 37° C. for 12 h. The minimum inhibitoryconcentration that suppressed the growth of at least 99% of the bacteria(MIC₉₉) was established based on culture turbidity. Additional pathogenswere grown and prepared similarly as above, with the exception ofoptimizing the growth media to match an organism's nutritionalrequirements (Streptococcus pyogenes, Todd Hewitt broth; Listeriamonocytogenes, Enterococcus faecalis st. U503 [VRE], and Staphylococcusaureus st. NRS384/USA300 [MRSA], brain heart infusion). Positivecontrols: S. pyogenes and L. monocytogenes, kanamycin; E. faecalis,tetracycline; S. aureus, vancomycin). Bactericidal activity wasdetermined by diluting 1 μL of B. anthracis strain Sterne grown with 8μg/mL PZN into 99 μL of media. The sample was then streaked onto LB agarplates and incubated for 24 h for counting colony-forming units.

Agar Diffusion Bioassay.

B. anthracis strain Sterne was grown as described previously and dilutedto OD₆₀₀ of 0.13. The diluted culture (100 μL) was inoculated onto an LBplate and allowed to dry. HPLC-purified PZN (50-200 μg) was added to apaper disk, dried, and added to the plate. Cultures were then incubatedat 37° C. for 12 h. Kanamycin (8-25 μg) was used as a positive control,and 80% MeCN was the negative (solvent) control.

Microscopy.

Differential interference contrast (DIC) microscopy images were obtainedby preparing live cell images of B. anthracis cultures. Samples werepretreated with or without PZN at 4 μg/mL (MIC₉₉), and morphology wasassessed using a Zeiss LSM 700 microscope. The objective used was aPlan-Apochromat 63×/1.40 Oil DIC M27. The analysis software used wasProgram Zen 2009 Light Edition.

On-Line RPLC-FTMS.

All reverse phase liquid chromatography (RPLC)-Fourier-transform massspectrometry (FTMS) was conducted using an Agilent 1200 high performanceLC(HPLC) system with an autosampler coupled directly to a ThermoFisherScientific LTQ-FT hybrid linear ion trap-FTMS system operating at 11tesla. The MS was calibrated weekly using the calibration mixture andinstructions specified by the manufacturer. All instrument parameterswere tuned according to the manufacturer's instructions (employingbovine ubiquitin for tuning purposes). For all analyses of PZN, a 1mm×150 mm Jupiter C18 column (Phenomenex, 300 Å, 5 μm) was connectedin-line with the electrospray ionization source (operated at ˜5 kV witha capillary temperature of 200-250° C.) for the MS system. A typicalsample was loaded onto the column using the autosampler and separatedusing a linear gradient of H₂O/MeCN and 0.1% formic acid with theanalytes eluted directly into the MS. All ionized species were subjectedto an MS method with five MS and MS/MS events: 1) full scan measurementof all intact peptides (all ions detected in the FTMS in profile mode;resolution: 100,000; m/z range detected: 400-2000), 2-5) data-dependentMS/MS on the first, second, third, and fourth most abundant ions fromscan (1) using collision induced dissociation (CID) (all ions detectedin the FTMS in profile mode; minimum target signal counts: 5,000;resolution: 50,000; m/z range detected: dependent on target m/z, defaultcharge state: 2, isolation width: 5 m/z, normalized collision energy(NCE): 35; activation q value: 0.40; activation time: 30 ms). During allanalyses, dynamic exclusion was enabled with the following settings:repeat count—2, repeat duration—30 s, exclusion list size—300, exclusionduration—60 s.

Direct Infusion FTMS.

After lyophilization for at least 24 h, HPLC purified samples weredissolved in 80% MeOH (to ˜0.5 mg/mL) and then further diluted 10-foldinto 50% MeOH supplemented with 0.1% formic acid. The diluted sampleswere directly infused using an Advion Nanomate 100. The singly chargedions were targeted for CID using identical settings as above, exceptthat the resolution was set to 100,000.

N-Terminal Labeling.

Purified PZN and desmethylPZN were dissolved in 80% MeCN, 10 mM MOPS (pH8.0) to a final concentration of 1.5 mM. An aliquot (5 μL) wastransferred to a microfuge tube containing 5 μL of 80% MeCN, 10 mM MOPS(pH 8.0) supplemented with 20 mM EZ-Link® sulfo-NHS-biotin. Controlreactions lacked the NHS-biotin reagent. The samples were allowed toreact for 3 h at 23° C. prior to analysis on an Applied BiosystemsVoyager DE-STR MALDI-TOF-MS.

NMR.

PZN was produced from low oxygenation cultures and purified as describedabove. PZN (700 μg) was dissolved in 200 μL of DMSO-d₆ and placed intoan Advanced Shigemi 5 mm NMR tube matched to DMSO-d₆. NMR experimentswere conducted on a Varian Unity Inova 500 NB (¹H-¹H-gCOSY) and a VarianUnity Inova 600 spectrometer (¹H, ¹H-¹H-TOCSY and ¹H-¹³C-gHMBC) using a5 mm Varian ¹H{¹³C/¹⁵N} PFG Z probe and 5 mm Varian ¹H{¹³C/¹⁵N} XYZ PFGtriple resonance probe, respectively. The ¹H-NMR, TOCSY and gHMBCexperiments were conducted at 25° C. and utilized water suppression. Amixing time of 150 ms was used for the TOCSY. For the gHMBC, ¹J and^(n)J were set to 140 and 8 Hz, respectively. Chemical shifts werereferenced using DMSO (δ_(H)=2.50 and δ_(C)=39.51), and the spectra wereprocessed and analyzed using MestReC. Stereochemical configuration wasassumed to be identical to the ribosomally produced precursor peptide.

Production of PZN from Bacillus pumilus ATCC 7061.

Cultures were prepared as described above for Bacillus amyloliquefaciensFZB42 cells, with the exception that no antibiotics were added. Themethod employed for metabolite extraction and HPLC purification wereidentical to samples from B. amyloliquefaciens. Purified fractions wereanalyzed on a Bruker Daltonics ultrafleXtreme MALDI-TOF/TOF instrumentoperating in reflector/positive mode. Sinapic acid was used as thematrix.

Results.

Mass spectrometry (MS) was used as the main spectroscopic tool inelucidating plantazolicin structure. Through the use of high-resolution,linear ion trap Fourier Transform hybrid MS (LTQ-FT) operating at 11tesla, the mass of the protonated form of PZN was measured to be1336.4783 Da (FIG. 1 a and FIG. 5). Due to the high mass accuracy ofFT-MS and the known sequence of the core region of the precursor peptide(₁RCTCTTIISSSSTF₁₄) (Lee et al., (2010) Proc Natl Acad Sci USA 105,5879-5884, Scholz et al., 2011, J Bacteriol 193, 215-224), the molecularformula of [PZN+H]⁺ could be deduced (C₆₃H₇₀N₁₇O₁₃S₂; theoreticalmonoisotopic mass=1336.4780 Da; error, 0.15 ppm). This formula requiredthat 9 out of 10 heterocyclizable residues (Cys, Ser, Thr) in the coreregion of the precursor peptide be converted to the azole heterocycle(FIG. 1A). Due to their adjacent positions, these processed residuesform a contiguous polyazole, which was supported by spectrophotometricanalysis. PZN gave absorption bands at 260 nm (λ_(max)), 310 nm (minorshoulder), and 370 nm (very weak shoulder), indicating the presence of acomplex chromophore (FIG. 6). The remaining heterocyclizable residue wasleft at the azoline (thiazoline, oxazoline, or methyloxazoline)oxidation state. Also, this formula required leader peptide cleavageafter Ala-Ala and two methylation events, consistent with earlierdeletion studies (Scholz et al., supra).

Collision induced dissociation (CID) was then used to localize thesite(s) of dimethylation and the azoline heterocycle. Analysis of thedoubly charged PZN ion using in-line HPLC-FTMS resulted in a spectrumthat was featureless from m/z ˜700-1100 as a result of contiguousheterocycle formation (FIG. 5). Nonetheless, the production of severaldiagnostic fragment ions was noted including peptide bond cleavage atIle-Ile. The masses of these resultant ions demonstrated that theN-terminal (b⁺ ion) fragment contained both posttranslational methylgroups and that the C-terminal (y⁺ ion) fragment contained the azoline(which was now restricted to either oxazoline or methyloxazoline due tothe absence of Cys on this fragment). Other informative fragment ionswere derived from cleavage between Arg1-Cys2(thiazole) andThr13(methyloxazoline)-Phe14. The former cleavage event demonstratedthat both posttranslational methyl groups were localized to Arg1.

Cleavage between Thr13-Phe14 led to the formation of severaldecomposition products that permitted the localization of the(methyl)oxazoline to Thr13. From the apparently unstable parent ion, aformal loss of allene from methyloxazoline (C₃H₄, 40.0313 Da) to yield aC-terminal amide was frequently observed (FIG. 5). Further support forthe location of the azoline heterocycle came from hydrolysis studies, asdiscussed below. Proposed structures for all assignable ions are givenin FIGS. 7 and 8.

Upon in-depth FTMS analysis of singly charged PZN, which was introducedby direct infusion, much larger ions relative to doubly charged PZNparent ions were routinely observed including the ones consistent withthe loss of guanidine (−59.0483 Da, m/z 1277.4299; error, 0.16 ppm)(FIG. 1B). This indicated that the site of dimethylation was restrictedto either the N-terminal amine or the alkyl sidechain of Arg1. Thelatter was thought to be highly improbable since the enzyme known tocatalyze this reaction (PznL) was predicted by sequence alignment to bea S-adenosylmethionine (SAM)-dependent methyltransferase. The onlySAM-dependent enzymes capable of engaging in C—H bond activation are theradical SAM enzymes, which are identifiable by numerous conserved Cysthat form Fe—S clusters, which are lacking in PznL (Leet et al., supra,Scholz et al., supra). Higher order CID was performed on thedeguanidinated form of PZN (m/z 1277), providing corroborating evidencefor N-terminal dimethylation (FIG. 9). In addition to CID analysis,further support for the N-terminus being the site of dimethylation inPZN came from chemoselective labeling.

HPLC-purified PZN and desmethylPZN (from the pznL methyltransferasedeletion strain) were reacted with the amine-specific reagent,N-hydroxysuccinimide (NHS)-biotin (Sholz et al., supra). As observed byMALDI-MS, labeling was only successful in the desmethylPZN reaction,indicating the presence of a free amine in this compound, but not in PZN(FIG. 10). From this, it has become clear that the leader peptidecleavage occurs before methylation, and that the ABC transport systemdoes not distinguish between PZN and desmethylPZN.

From the apparent hydrolysis of PZN following SDS-PAGE, it was shownthat PZN contained an azoline. Such heterocycles are prone to both acid-and base-catalyzed hydrolysis (Frump, J. A. (1971) Chemical Reviews 71,483; Martin et al., Journal of the American Chemical Society 83,4835-4838). Mild acid treatment of PZN yielded m/z 1354 (+18), which wasshown by CID studies to be from the reconstitution of the Thr13 residueof the precursor peptide (FIG. 1B). Higher order tandem MS experimentsfurther confirmed the location of the PZN methyloxazoline moiety (FIGS.8 and 9). It is interesting to note that this methyloxazoline was thesole heterocycle not processed by the TOMM dehydrogenase, suggestingthat the FMN-dependent dehydrogenase (B) is capable of distinguishingthis heterocycle from others during the biosynthetic process. During theextensive MS analysis of PZN, it was noticed that fragmentation of themethyloxazoline moiety gave rise to a characteristic mass loss. CIDfragmentation of PZN yielded an intense daughter ion of m/z 1292.4519(FIG. 1B). The mass difference from the PZN parent ion was 44.0261 Da,which was consistent with the neutral loss of acetaldehyde (C₂H₄O, exactmass=44.0262). Loss of acetaldehyde is conceivable fromcyclo-elimination of methyloxazoline to yield a nitrile ylide, which canre-cyclize to form an azirine. The microscopic reverse of this reactionpathway is well known in the chemical literature where azirines arereacted with aldehydes to form oxazolines via 1,3-dipolar cycloaddition(Frump, J. A. supra, Giezenda et al., 1973, Helvetica Chimica Acta 56,2611-2627; Sa et al., 1996, Journal of Organic Chemistry 61, 3749-3752).Of note, the loss of acetaldehyde was observed only when methyloxazolinewas present on the parent ion (see FIG. 1B-1C and FIGS. 7-9).

To corroborate the proposed structure elucidated by MS, a series oftwo-dimensional NMR experiments was performed, including ¹H-¹H-gCOSY,¹H-¹H-TOCSY, and ¹H-¹³C-gHMBC on a 600 MHz instrument (FIGS. 11-13).Briefly, the gCOSY and TOCSY spectra confirmed the following: i. due tothe absence of NH and C_(α)H correlations, all Cys, Ser, and Thr wereheterocyclized (the NH and C_(α)H correlations were readily visible forall internal residues with an intact amide bond—Ile, Ile, Phe); ii. thecarbon framework of the Arg1, Ile7, Ile8, and Phe14 side chains were notmodified and, iii. the sole azoline moiety of PZN occurred on a Thr. The¹H-¹³C-gHMBC spectrum further validated findings from the ¹H-¹Hexperiments, in addition to proving the methylation sites asN^(α),N^(α)-dimethylArg (FIG. 13). N-terminal methylation of ribosomallyproduced peptides in bacteria is an exceedingly rare posttranslationalmodification. While N-terminal dimethylation has been described on Ala(e.g. cypemycin), N^(α),N^(α)-dimethylArg appears to be a novelposttranslational modification (Garavelli, J. S. 2004, Proteomics 4,1527-1533).

During the course of optimizing the production of PZN for detailedspectroscopic analysis, it was noticed that the level of cultureoxygenation had an impact on the production of PZN and derivativemetabolites. Under low oxygen fermentation, PZN (m/z 1336) was themajority species present after a non-lytic, cell surface extractionprocedure, as demonstrated by the UV trace, total ion chromatogram(TIC), and the extracted ion chromatogram (EIC, FIG. 2 a-c). The productof methyloxazoline ring opening (i.e. hydrolyzed PZN, m/z 1354) was alsomonitored (FIG. 2C-2D). The m/z 1338 species that “coeluted” with 1336at 19.9 min was actually the second isotope peak (two extra neutrons) ofm/z 1336 (FIG. 14).

Under oxygen saturated cultivation, UV and TIC monitoring revealed anadditional, highly abundant species at 14.7 min (FIG. 2A-2B). MSanalysis demonstrated this species was m/z 1338, suggestive of a reducedPZN species (dihydroPZN) containing two azoline heterocycles (FIG. 2D,FIG. 15). The earlier elution time on reverse-phase chromatographysuggested that this species was more polar than PZN, which wasconsistent with the replacement of an aromatic azole with a protonatedazoline (expected in 0.1% formic acid). After treatment of m/z 1338 withmild aqueous acid, two additions of water were observed (m/z 1356 and1374). Tandem MS was then used to demonstrate that the second azolinesite was located on the N-terminal half of PZN (FIG. 16). Higher orderCID analysis prompted the neutral loss of acetaldehyde, indicating thatthe second azoline heterocycle was derived from Thr, likely the residuedirectly preceding Ile (Thr6, data not shown). To an approximation, thisposition was sterically and electronically equivalent to the previouslydiscussed methyloxazoline (Thr13) since both lie between an N-terminaltetra-azole and a C-terminal unmodified, hydrophobic residue (FIG. 1A).

The production of additional PZN-related species was also observed fordesmethylPZN when oxygenation levels were increased during cultivationof the pznL methyltransferase deletion strain (FIGS. 17-18). As above,the pznL deletion strain only produced desmethylPZN (m/z 1308) under lowoxygenation. Under oxygen-saturated conditions, a significant amount ofan earlier eluting species, dihydrodesmethylPZN (m/z 1310) wasgenerated. It is possible that azoline oxidation, through the action ofthe FMN-containing dehydrogenase, was the rate-determining step in PZNbiosynthesis. While not being bound to a particular theory, it may bepossible, that with increased aeration (faster metabolism), partiallyprocessed PZN products are more rapidly produced and accepted assubstrates by proteins acting downstream of the dehydrogenase (i.e. theleader peptidase, methyltransferase, and ABC transport system). Thebiosynthetic implication of obtaining PZN oxidation intermediates isthat the rate of methyloxazoline oxidation at “Thr6” (putative) andThr13 (FIG. 1A) is slower than the dissociation rate from theheterotrimeric synthetase (BCD) complex and subsequent maturation steps.During native PZN biosynthesis, it is most probable that “Thr6” is thelast position to be oxidized. From this observed PZN oxidationintermediate, it becomes apparent that cyclodehydration precedesdehydrogenation, as has been previously supported by in vitroreconstitution experiments but never before demonstrated in vivo(McIntosh and Schmidt, 2010, Chembiochem 11, 1413-1421; Milne et al.,1999, Biochemistry 38, 4768-4781).

It was determined that PZN exhibited activity primarily towards Bacillussp., including B. subtilis. PZN exhibited no activity against any testedGram-negative organisms. To further define the selectivity within theGram-positive organisms, the scope of PZN activity was evaluated towardsa panel of ubiquitous human pathogens, including methicillin-resistantStaphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis(VRE), Listeria monocytogenes, Streptococcus pyogenes, and Bacillusanthracis strain Sterne (a surrogate for the BSL3 pathogen, which lacksthe poly-D-glutamic acid capsule). Using a microbroth dilution bioassay,potent growth inhibition of B. anthracis was observed (FIG. 3A). Allother species were unaffected by PZN, with the exception of S. pyogenes,which was only inhibited by very high concentrations of PZN.

The specificity for PZN against B. anthracis was recapitulated in anagar diffusion bioassay (FIG. 3B), as inhibition zones were not observedfor any other tested bacterium (data not shown). The action of PZN uponB. anthracis was bactericidal, as reculturing of treated cells in theabsence of PZN led to no bacterial growth. Live cell imaging(non-stained, non-fixed) by differential interference contrast (DIC)microscopy revealed that B. anthracis treated with PZN at 4 μg/mLunderwent massive lysis, as evidenced by an abundance of cellular debris(data not shown). Of the few remaining cells that survived, markedchanges were observed in the appearance of the cell surface (FIG.3C-3D). While not being bound to a particular theory, it is believedthat PZN either directly or indirectly disrupts peptidoglycanbiosynthesis leading to the cell wall becoming structurally compromised.

Dimethylation of the α-amino group was apparently important for PZN'santibiotic activity, as desmethylPZN was devoid of activity against B.anthracis in both bioassays (FIG. 3A). While the molecular basis forthis effect is not currently known, dimethylation increases aminebasicity, increases lipophilicity, and removes two potential H-bonddonors. Also tested were the effects of hydrolyzing the methyloxazolinemoiety of PZN (hydrolyzed PZN, m/z 1354) and the variant with twoazolines (dihydroPZN, m/z 1338). While hydrolyzed PZN retainedmeasurable activity towards B. anthracis, dihydroPZN was devoid ofactivity. Due to difficulty in separating dihydroPZN from the mono- anddi-hydrolyzed forms (m/z 1356 and 1374), these bioassays were performedusing a 1:2:2 mixture (non:mono:di). The lack of activity with thismixture of hydrolyzed, dihydroPZN compounds might be attributable to thefact that hydrolyzed PZN is roughly 8-fold less active than PZN (FIG. 3a). It is also possible that the production of dihydroPZN is an artifactof laboratory cultivation.

A targeted bioinformatics survey using the thiazole/oxazole synthetaseproteins (cyclodehydratase, dehydrogenase, and docking protein) of PZNyielded four highly related biosynthetic gene clusters (FIG. 4). Thecluster found in Bacillus pumilus ATCC 7061 (also a plant-growthpromoting saprophyte) was of identical gene order and direction as thecluster from B. amyloliquefaciens FZB42. The remaining three PZN-likebiosynthetic clusters were found in the Actinobacteria phylum includingClavibacter michiganensis subsp. sepedonicus (potato pathogen) (30),Corynebacterium urealyticum DSM 7109 (human skin-associated bacterium,causative agent of some urinary tract infections) and Brevibacteriumlinens BL2 (human skin-associated bacterium). The PZN cassettes from C.urealyticum and B. linens have amino acid similarity values much higherwith each other than the other PZN producers (FIG. 19). In each of thefive species, the PZN biosynthetic cluster contained the canonical TOMMgenes: a precursor peptide, dehydrogenase, cyclodehydratase, and dockingprotein. Beyond this, all five clusters also include a putativemembrane-spanning leader peptidase from the type II CAAX superfamily(31), SAM-dependent methyltransferase, and a required protein of unknownfunction. Conversely, homologs of the PznF immunity protein and PznGHtransporters were not found in the local genomic context for the PZNbiosynthetic gene clusters for C. urealyticum and B. linens (FIG. 4 a).This suggests a distinct mechanism of immunity and chromosomally distanttransporters for these PZN variants. Alternatively, the PZNs from C.urealyticum and B. linens could act intracellularly or the biosyntheticgene cluster might always be silent (non-product forming).

Based on the identical amino acid sequence of the core regions of theprecursor peptides from FZB42 and B. pumilus, it would have beenexpected that these species produced identical compounds (FIG. 4B). Todirectly test if B. pumilus was indeed producing PZN, stationary phaseB. pumilus ATCC 7061 cultures were cell surface extracted in anidentical manner as with FZB42. MALDI-TOF-MS of HPLC-purified fractionsrevealed the presence of m/z 1336, and in an earlier fraction, m/z 1354(+H₂O), supporting the production of PZN and hydrolyzed PZN from thisstrain (FIG. 20 a-b). The identity of this species as PZN was confirmedby high accuracy mass measurement (LTQ-FT-MS) and CID analysis (FIG. 20c-e). As anticipated, B. amylo. FZB42 and B. pumilus ATCC 7061 were notsusceptible to the action of PZN (no observable inhibition at 128μg/mL). A non-plant associated strain of B. amylo. (NRRL B-14393), whichdoes not produce PZN, was also completely resistant (data not shown).Resistance within the Bacillus genus to PZN is clearly complex, with afew strains being bona fide PZN producers and others simply harboringthe immunity gene [e.g. B. amylo. strains YAU-Y2 and NAU-B3 and B.atrophaeus 1942, BATR1942_(—)01200, 94% identical to FZB42] (Scholz etal., supra). Early attempts to isolate a PZN-type natural product fromthe Actinobacteria family members were not successful. The lack of asignal by MALDI-MS and reverse transcriptase-PCR suggested that thebiosynthetic genes were not transcribed under tested cultivationconditions (data not shown). As with many “silent” gene clusters, highlyprecise environmental conditions may be necessary for the bacterium toproduce particular natural products.

Sequence alignment of all five PZN precursor peptides showed that therehas been evolutionary pressure to maintain a nearly invariant chemotypegiving rise to the PZN structure (from N- to C-terminus): leader peptidecleavage site and N-terminal Arg (FEPxAA*R), five cyclizable residueswith position 2 and 4 always Cys and position 6 always Thr, twohydrophobic residues, five cyclizable residues, and a more variableC-terminus that ends with Phe, Trp-Gly, or Gly-Gly (FIG. 4B).

When introducing elements of the present invention or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above products and methodswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

1. A plantazolicin-like compound having the structure:(X¹)—(X²)₅—(X³)₂—(X⁴)₅—(X⁵)_(n) or a pharmaceutically acceptable salt orester thereof, wherein X¹ is

R¹ and R² are each independently hydrogen or lower alkyl; each X² isindependently an azole; each X³ is independently a hydrophobic aminoacid; each X⁴ is independently an azole or azoline; and each X⁵ isindependently an amino acid, wherein n is 1 or
 2. 2. The compound ofclaim 1 wherein the compound includes at least one of the following: R¹and R² are each independently hydrogen or methyl; each X² isindependently selected from the group consisting of: pyrazole,imidazole, thiazole, oxazole, isoxazole, isothiazole, pyrrole, triazole,tetrazole, and pentazole; each X³ is independently selected from thegroup consisting of alanine (Ala), valine (Val), isoleucine (Ile),leucine (Leu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr) andtryptophan (Trp); each X⁴ is independently an azole selected from thegroup consisting of pyrazole, imidazole, thiazole, oxazole, isoxazole,isothiazole, pyrrole, triazole, tetrazole, and pentazole, or an azolineselected from the group consisting of pyrazoline, imidazoline,thiazoline, oxazoline, isoxazoline, isothiazoline, pyrroline,triazoline, tetrazoline, and pentazoline; each X⁵ is independentlyselected from the group consisting of phenylalanine, tyrosine andtryptophan; or n is 1 or
 2. 3. (canceled)
 4. The compound of claim 2wherein each X² is independently thiazole or oxazole; each X³ isindependently isoleucine, phenylalanine or tryptophan; each X⁴ isindependently thiazole, oxazole or oxazoline; or X⁵ is phenylalanine.5.-12. (canceled)
 13. The compound of claim 1 wherein the compound isplantazolicin having the structure:

or a pharmaceutically acceptable salt or ester thereof.
 14. Apharmaceutical composition comprising the compound of claim 13 and apharmaceutically acceptable carrier.
 15. The pharmaceutical compositionof claim 14, wherein the composition comprises from about 100 μg toabout 100 mg of the compound.
 16. The pharmaceutical composition ofclaim 14, wherein the composition comprises from about 1 mg to about 50mg of the compound.
 17. (canceled)
 18. A method for treating orpreventing a Bacillus anthracis infection or a Bacillus cereusinfection, the method comprising administering a pharmaceuticalcomposition of claim 14 to an animal subject in need thereof.
 19. Themethod of claim 18, wherein the animal subject is infected with anthraxspores and has anthrax disease.
 20. The method of claim 18, wherein theanimal subject is a human.
 21. The method of claim 18, wherein theanimal subject is a dog, cat, horse, sheep, goat, or cow.
 22. The methodof claim 18, wherein the anthrax is inhalation anthrax, cutaneousanthrax, oropharyngeal anthrax or gastrointestinal anthrax.
 23. Themethod of claim 18, wherein the composition is administered orally,intravenously, intramuscularly or subcutaneously. 24.-25. (canceled) 26.The method of claim 18, wherein the composition is administered once,twice or three times a day. 27.-32. (canceled)
 33. A method foridentifying a plantazolicin-like protein, wherein the method comprises:identifying a bacterial amino acid sequence exhibiting at least 50%amino acid identity to a plantazolicin precursor peptide from Bacillusamyloliquefaciens FZB42; obtaining a post-translationally modifiedproduct of the bacterial amino acid sequence; and testing thepost-translationally modified product of the bacterial amino acidsequence in a Bacillus anthracis growth inhibitory assay, whereinability to inhibit the growth of Bacillus anthracis indicates that thebacterial amino acid sequence encodes a plantazolicin-like protein.34.-35. (canceled)
 36. A method for producing the plantazolicin compoundof claim 13, the method comprising: growing Bacillus cells in culture;collecting the Bacillus cells, thereby obtaining the harvested Bacilluscells; obtaining a crude plantazolicin extract from the harvestedBacillus cells; and purifying the plantazolicin compound from the crudeplantazolicin extract, wherein the Bacillus cells are Bacillusamyloliquefaciens FZB42 cells or Bacillus pumilus cells.
 37. (canceled)38. The method of claim 36, wherein growing cells in culture isperformed under low oxygenation conditions; collecting the cells isperformed by centrifugation; obtaining the crude plantazolicin extractis performed by methanolic extraction; or purifying the plantazolicincompound is performed by high performance liquid chromatography (HPLC).39.-42. (canceled)
 43. The method of claim 36, further comprising thestep of converting the purified plantazolicin compound to a salt or anester thereof.
 44. A method of identifying Bacillus anthraciscomprising: combining the compound of claim 1 with a biological samplesuspected of containing a human pathogen to form a mixture; andanalyzing the mixture to determine the extent of cell lysis or cellgrowth inhibition, wherein growth inhibition or lysis of a majority ofcells in the sample is indicative of the presence of Bacillus anthracis.45. The method of claim 44 wherein the compound is the plantazolicincompound of claim
 13. 46. The method of claim 44 wherein the extent ofcell lysis is analyzed wherein lysis of a majority of cells in thesample is indicative of the presence of Bacillus anthracis.
 47. Themethod of claim 46 wherein the mixture is analyzed via live cellimaging.
 48. The method of claim 47 wherein the live cell imaging isdifferential interference contrast (DIC) microscopy.
 49. The method ofclaim 48 wherein the mixture is analyzed to determine the extent of cellgrowth inhibition, wherein growth inhibition is indicative of thepresence of Bacillus anthracis.
 50. The method of claim 49 wherein theextent of growth inhibition of the cultured mixture is analyzed via amicrobroth dilution assay.
 51. The method of claim 49 further comprisingculturing the mixture prior to analyzing the extent of growth inhibitionof the cultured mixture via an agar disk diffusion assay.
 52. The methodof claim 49 wherein the extent of growth inhibition of the mixture isanalyzed by visual observation of a clear zone in the mixture if growthinhibition has occurred.